Cultures of and methods of manufacturing squamous cell carcinoma cells

ABSTRACT

The present disclosure generally relates to a cell culturing system, and specifically to a three-dimensional cell culturing system for modified primary keratinocytes that mimic those cells of in vivo squamous cell carcinoma cells. The three-dimensional cell culturing system allows for flexibility in size and modularity of skin cancer cell growth with physiologically-relevant results.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional Application No. 62/937,787, filed Nov. 19, 2019, the entirety of which is hereby incorporated by reference for all purposes.

FIELD OF THE DISCLOSURE

The present disclosure generally relates to a method of generating genome-edited keratinocytes, a cell culturing system, and particularly to a cell culturing system for genome-edited keratinocytes that mimic those cells of in vivo squamous cell carcinoma cells and their progenitors. The cell culturing system of the present disclosure allows for flexibility in size and modularity of cancer cell growth with physiologically-relevant results.

BACKGROUND

The epidermis is a stratified squamous epithelium that forms the protective covering of the skin. Only the innermost, basal layer of epidermal cells typically has the capacity for DNA synthesis and mitosis. During epidermal differentiation, a basal cell will begin its journey to the skin surface. In transit, it undergoes a series of morphological and biochemical changes that culminate in the production of dead, flattened, enucleated squames, which are sloughed from the surface, and continually replaced by inner cells differentiating outward. A representative diagram of intralayers in epithelium tissue is shown in FIG. 1 .

Squamous cell carcinoma (SCC) is common form of cancer and is the second most common form of skin cancer in the United States. SCCs can affect any of the squamous cells that form the surface of the skin, line of hollow organs in the body, and line the respiratory and digestive tracts. Common types of this cancer include squamous cancers of the skin, upper aerodigestive tract, esophagus, vagina, cervix, anus, bladder, and lung. Incidence and prognosis varies by primary organ.

The evolution and progression of SCC are thought to result from multiple stepwise alterations of cellular and molecular pathways in the squamous epithelium. Mounting evidence suggests a model of molecular progression from premalignant lesions to invasive disease (FIG. 2 ).

Squamous cell carcinoma of the skin (or skin SCC) is a common form of skin cancer that develops in the squamous cells that make up the middle and outer layers of the skin. Skin SCC affects 660,000 people per year and generally carries an excellent prognosis. For a very small skin SCC that has a low risk of spreading, less invasive treatments, including curettage and electrodessication (C and E), laser therapy, freezing, and photodynamic therapy, may be used. For a larger skin SCC that extends deeper into the skin, more invasive treatments, such as simple excision, mohs surgery, or radiation therapy, are recommended. For a skin SCC that spreads beyond the skin, drug treatments, including chemotherapy, targeted drug therapy, and immunotherapy, are recommended.

Currently, there are only two drugs have been approved specifically for treatment of SCCs. Moreover, for the upper aerodigestive tract, SCC is by far the most common malignancy and the median five-year survival rate for this type of SCC is approximately 50% and has only improved minimally over the past 50 years. Thus, additional therapeutics and treatments are still needed for squamous cell carcinomas, particularly for those that are not ineligible for surgical interventions and/or unresponsive to radiotherapy and chemotherapy.

SUMMARY OF THE EMBODIMENTS

In one aspect, the present disclosure relates to a method of genetically altering one or a plurality of primary keratinocytes comprising exposing the one or plurality of primary keratinocytes to one or a plurality of mutagens for a time period sufficient to mutate one or a plurality of wild-type genes such that one or a plurality of genome-edited primary keratinocytes deficient in expression of the one or plurality of wild-type genes are produced. In some embodiments, the one or plurality of wild-type genes being mutated are chosen from Table 1 provided in the present disclosure.

In some embodiments, the one or plurality of mutagens is a vector or a gene-modifying enzyme. In some embodiments, the gene-modifying enzyme is a Cas protein, a meganuclease, a transcription activator-like effector nucleases (TALEN), a transposon, a zinc-finger nuclease (ZFN), or a recombinase.

In some embodiments, the method of the present disclosure further comprises transfecting the one or plurality of primary keratinocytes with a plasmid comprising a nucleic acid sequence encoding the one or plurality of mutagens. In some embodiments, the nucleic acid sequence encodes a Cas9 protein. In some embodiments, the plasmid further comprises one or a plurality of small guide RNAs guiding the Cas9 protein to the one or plurality of wild-type genes for genome editing.

In some embodiments, the method of the present disclosure further comprises a step of selecting the one or plurality of genome-edited primary keratinocytes deficient in expression of the one or plurality of wild-type genes. In some embodiments, the step of selecting is performed based on expression of a marker gene. In some embodiments, the marker gene is a fluorescent reporter gene, an antibiotic resistance gene, or a surface protein-coding gene. In some embodiments, the fluorescent reporter gene encodes a green fluorescent protein (GFP), a tdTomato fluorescent protein, an EBFP2 fluorescent protein, a mKate2 fluorescent protein, or a mKO fluorescent protein. In some embodiments, the antibiotic resistance gene is an ampicillin resistance gene, a kanamycin resistance gene, or a puromycin resistance gene. In some embodiments, the surface protein-coding gene encodes a kinase domain deleted low-affinity NGF receptor (DeltaLNGFR) coupled with a streptavidin binding peptide (SBP).

In some embodiments, the one or plurality of genome-edited primary keratinocytes are deficient in expression of at least one of wild-type TP53, CDKN2A, and NOTCH1. In some embodiments, the one or plurality of genome-edited primary keratinocytes are deficient in expression of wild-type TP53, CDKN2A, and NOTCH1.

In another aspect, the present disclosure relates to a method of generating a population of one or a plurality of genome-edited keratinocytes comprising:

-   -   a) generating and selecting one or a plurality of genome-edited         primary keratinocytes deficient in expression of one or a         plurality of wild-type genes according to any of the embodiments         disclosed herein;     -   b) expanding the one or plurality of genome-edited primary         keratinocytes into the population of one or plurality of         genome-edited keratinocytes.

In some embodiments, the one or plurality of primary keratinocytes used in any of the methods disclosed herein are primary human keratinocytes.

In a further aspect, the present disclosure relates to a composition comprising the population of one or plurality of genome-edited keratinocytes generated by any of the methods disclosed herein. In some embodiments, the population of one or plurality of genome-edited keratinocytes is deficient in expression of one or a plurality of wild-type genes chosen from Table 1. In some embodiments, the population of one or plurality of genome-edited keratinocytes is deficient in expression of at least one of wild-type TP53, CDKN2A, and NOTCH1. In some embodiments, the population of one or plurality of genome-edited keratinocytes is deficient in expression of wild-type TP53, CDKN2A, and NOTCH1.

In yet another aspect, the present disclosure relates to a composition comprising one or a plurality of genome-edited keratinocytes that is deficient in expression of one or a plurality of wild-type genes chosen from Table 1. In some embodiments, the population of one or plurality of genome-edited keratinocytes is deficient in expression of at least one of wild-type TP53, CDKN2A, and NOTCH1. In some embodiments, the one or plurality of genome-edited keratinocytes are deficient in expression of wild-type TP53, CDKN2A and NOTCH1. In some embodiments, the keratinocytes are primary human keratinocytes.

In some embodiments, the disclosure relates to a composition comprising one or a plurality of cells, particularly keratinocytes, comprising one or a plurality of mutations in a gene comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 307. In some embodiments, the disclosure relates to a composition comprising one or a plurality of cells, particularly keratinocytes, comprising one or a plurality of mutations in a gene comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 308. In some embodiments, the disclosure relates to a composition comprising one or a plurality of cells, particularly keratinocytes, comprising one or a plurality of mutations in a gene comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 309. In some embodiments, the disclosure relates to a composition comprising one or a plurality of cells, particularly keratinocytes, comprising one or a plurality of mutations in a gene comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 307. In some embodiments, the disclosure relates to a composition comprising one or a plurality of cells, particularly keratinocytes, comprising one or a plurality of mutations in each of the genes comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 307 and SEQ ID NO: 308. In some embodiments, the disclosure relates to a composition comprising one or a plurality of cells, particularly keratinocytes, comprising one or a plurality of mutations in each of the genes comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 307 and SEQ ID NO: 309. In some embodiments, the disclosure relates to a composition comprising one or a plurality of cells, particularly keratinocytes, comprising one or a plurality of mutations in each of the genes comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 308 and SEQ ID NO: 309. In some embodiments, the disclosure relates to a composition comprising one or a plurality of cells, particularly keratinocytes, comprising one or a plurality of mutations in each of the genes comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 307, SEQ ID NO: 308, and SEQ ID NO: 309.

In some embodiments, the disclosure relates to a composition comprising one or a plurality of cells, particularly keratinocytes, comprising one or a plurality of mutations in a gene encoding a polypeptide comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 347. In some embodiments, the disclosure relates to a composition comprising one or a plurality of cells, particularly keratinocytes, comprising one or a plurality of mutations in a gene encoding a polypeptide comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 348. In some embodiments, the disclosure relates to a composition comprising one or a plurality of cells, particularly keratinocytes, comprising one or a plurality of mutations in a gene encoding a polypeptide comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 349. In some embodiments, the disclosure relates to a composition comprising one or a plurality of cells, particularly keratinocytes, comprising one or a plurality of mutations in each of the genes encoding polypeptides comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 347 and SEQ ID NO: 348. In some embodiments, the disclosure relates to a composition comprising one or a plurality of cells, particularly keratinocytes, comprising one or a plurality of mutations in each of the genes encoding polypeptides comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 347 and SEQ ID NO: 349. In some embodiments, the disclosure relates to a composition comprising one or a plurality of cells, particularly keratinocytes, comprising one or a plurality of mutations in each of the genes encoding polypeptides comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 348 and SEQ ID NO: 349. In some embodiments, the disclosure relates to a composition comprising one or a plurality of cells, particularly keratinocytes, comprising one or a plurality of mutations in each of the genes encoding polypeptides comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 347, SEQ ID NO: 348 and SEQ ID NO: 349.

In some embodiments, the composition of the present disclosure further comprises a top layer of cells and a bottom layer of tissue, the bottom layer of tissue comprising extracellular matrix protein and one or multiple cell types and the top layer of cells comprising the one or plurality of genome-edited keratinocytes. In some embodiments, the cells of the bottom layer of tissue comprises one or a plurality of fibroblasts. In some embodiments, the cells of the bottom layer of tissue comprise one or a plurality of a combination of cells including fibroblasts, endothelium, CD45+ immune cells, pericytes, and/or myoepithelial cells. In some embodiments, the composition of the present disclosure further comprises a hydrogel layer positioned underneath the bottom layer of tissue. In some embodiments, the hydrogel layer comprises one or a plurality of growth factors or functional fragments thereof. In some embodiments, the one or plurality of growth factors are chosen from Table 4 provided in the present disclosure or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the growth factors listed in Table 4 of the present disclosure. In some embodiments, the hydrogel is Matrigel®.

In some embodiments, the composition of the present disclosure is at 37 degrees Celsius and exposed to about 5% CO2 for no less than 24 or 48 hours.

In some embodiments, the composition of the present disclosure further comprises at least a single insert comprising a biocompatible polymer and an inner wall defining an inner volume divided into an upper chamber and a bottom chamber, the upper chamber and bottom chamber positioned adjacently to one another and in fluid communication and separated by a ledge; the ledge positioned along an inner wall of the insert and defining a planar interface between the upper and bottom chambers. In some embodiments, the composition comprises a top layer of cells, a bottom layer of tissue and a hydrogel; each of the top layer of cells and bottom layer of tissue positioned in the upper chamber; and wherein the hydrogel is positioned in the bottom chamber; wherein the bottom layer of tissue comprises extracellular matrix protein and the top layer of cells comprises the one or plurality of genome-edited keratinocytes keratinocytes disclosed herein. In some embodiments, the top layer of cells comprises comprise one or a plurality of cells including fibroblasts, endothelial cells, CD45+ immune cells, pericytes, melanocytes. Merkel cells, and Langerhans cells, and/or myoepithelial cells. In some embodiments, the composition comprises at least about 2, 6, 12, 24, 48, 96, 192 or more inserts. In some embodiments, the composition comprises at least about 2, 6, 12, 24, 48, 96, 192 or more inserts, each insert comprising a space or opening or interface at the top of the insert separating the insert from a point exterior to the insert. In some embodiments, the interface is a planar or substantially planar opening with a surface area from about 0.1 to about 5 square centimeters. In some embodiments, the upper chamber has a volume from about 0.1 to about 9 centimeters cubed. In some embodiments, the bottom chamber has a volume from about 0.1 to about 9 centimeters cubed.

In some embodiments, the composition of the present disclosure is frozen at from about −20 to about −212 degrees Celsius.

In another aspect, the present disclosure relates to a system comprising:

-   -   a) at least one solid support comprising a bottom surface and         sidewalls, the bottom surface and sidewalls defining a culture         volume;     -   b) at least one insert as defined herein positioned on the         bottom surface of the at least one solid support; and     -   c) one or a plurality of keratinocytes that is deficient in         expression of one or a plurality of wild-type genes chosen from         Table 1 positioned within the upper chamber of the insert.         In some embodiments, the one or plurality of keratinocytes are         deficient in expression of at least one of wild-type TP53,         CDKN2A, and NOTCH1. In some embodiments, the one or plurality of         keratinocytes are deficient in expression of wild-type TP53,         CDNK2A, and NOTCH1. In some embodiments, the one or plurality of         keratinocytes are deficient in expression of wild-type TP53,         CDKN2A, and NOTCH1 in combination with mutations in other genes.         In some embodiments, the keratinocytes are primary human         keratinocytes.

In some embodiments, the system of the present disclosure further comprises a top layer of cells and a bottom layer of tissue, the bottom layer of tissue comprising extracellular matrix protein and the top layer of cells comprising the one or plurality of keratinocytes. In some embodiments, the bottom layer of tissue comprises one or a plurality of fibroblasts. In some embodiments, the system of the present disclosure further comprises a hydrogel layer positioned adjacently underneath the bottom layer of tissue. In some embodiments, the hydrogel layer comprises one or a plurality of growth factors or functional fragments thereof. In some embodiments, the one or plurality of growth factors are chosen from Table 4 provided in the present disclosure or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the growth factors listed in Table 4 of the present disclosure. In some embodiments, the hydrogel is Matrigel®.

In some embodiments, the system of the present disclosure further comprises an incubator in which the solid support is positioned and wherein the incubator comprises a heating element and an air inlet through which air content is controlled. In some embodiments, the temperature is about 37 degrees Celsius and comprises ambient air in contact with the solid support that comprises about 5% carbon dioxide.

In some embodiments, the system of the present disclosure further comprises culture media in the volume of the solid support with a cell culture media volume sufficient to cover the solid support but exposing one or plurality of keratinocytes to air. In some embodiments, the system of the present disclosure further comprises a tissue culture medium at a volume sufficient to cover the bottom tissue and create an air liquid interface immediately adjacent to or below the top layer of cells, such that the top layer of cells is exposed to air. In some embodiments, the air is filtered or sterilized in a closed system.

In some embodiments, the solid support in the system of the present disclosure comprises one or a plurality of wells or inserts from about 3 to about 5 centimeters in height. In some embodiments, the system comprises a solid support that is from about 1 to about 10 centimeters in height.

In some embodiments, the system of the present disclosure comprises a top layer of cells, a bottom layer of tissue and a hydrogel; each of the top layer of cells and bottom layer of tissue positioned in the upper chamber; and wherein the hydrogel positioned in the bottom chamber; wherein the bottom layer of tissue comprises extracellular matrix protein and the top layer of cells comprises the one or plurality of primary keratinocytes. In some embodiments, the system of the present disclosure comprises at least about 6, 12, 24, 48, 96, 192 or more inserts.

In some embodiments, the interface in the system of the present disclosure is a planar or substantially planar opening with a surface area from about 0.1 to about 5 square centimeters. In some embodiments, the upper chamber in the system of the present disclosure has a volume from about 0.1 to about 9 centimeters cubed. In some embodiments, the bottom chamber in the system of the present disclosure has a volume from about 0.1 to about 9 centimeters cubed.

In some embodiments, the system of the present disclosure further comprises a tissue culture medium at a volume sufficient to cover the bottom tissue and create an air liquid interface immediately adjacent to or below the top layer of cells, such that the top layer of cells is exposed to air. In some embodiments, the air is filtered or sterilized in a closed system.

In some embodiments, the system of the present disclosure further comprises a gas reservoir in fluid communication with the solid support via one or a plurality of gas lines; and a heating element operably positioned at or proximate to the solid support, such that the temperature of ambient air around the top layer of cells is from about 32 to about 42 degrees Celsius.

In some embodiments, the system of the present disclosure further comprises one or more therapeutic compounds.

In another aspect, the present disclosure provides a method of isolating a primary keratinocytes from skin tissue or mucosa comprising culturing a primary keratinocyte to culture medium while simultaneously exposing one or more keratinocytes to one or a plurality of tissue samples comprising extracellular matrix (ECM). In some embodiments, the skin tissue is obtained from any part of a human body. In some embodiments, the skin is anal skin. In some embodiments, the mucosa is oral mucosa, mucosa of the upper aerodigestive tract, esophageal mucosa, vaginal mucosa, or cervical mucosa.

In a further aspect, the present disclosure relates to a method of culturing a primary keratinocyte comprising exposing the primary keratinocyte to a system comprising:

-   -   a) at least one solid support comprising a bottom surface and         sidewalls, the bottom surface and sidewalls defining a culture         volume; and     -   b) at least one insert as defined herein positioned on the         bottom surface of the at least one solid support; wherein the         insert comprises a layer of tissue comprising ECM onto which the         primary keratinocytes are positioned.

In some embodiments, the step of exposing further comprises exposing the primary keratinocyte to cell culture media comprising:

a) DMEM/HAM F12(3:1) supplemented with 10% FBS;

b) 1% penicillin/streptomycin;

c) 1% antibiotic-antimycotic;

d) 3 μg/mL of adenine;

e) 3 ng/mL of cholera toxin; 40 ng/mL of hydrocortisone;

g) 5 μg/mL of insulin;

h) 10 ng/mL of EGF;

i) 5 μg/mL of Transferrin;

j) 1.5 ng/mL of triiodo-L-thyronine; and

k) 10 ng/mL of ciprofloxacin hydrochloride,

In some embodiments, the step of exposing further comprises exposing the primary keratinocyte to cell culture media comprising:

a) DMEM/HAM F12(3:1) supplemented with about 10% FBS;

b) about 1% penicillin/streptomycin;

c) about 1% antibiotic-antimycotic;

d) about 3 μg/mL of adenine;

e) about 3 ng/mL of cholera toxin;

about 40 ng/mL of hydrocortisone;

g) about 5 μg/mL of insulin;

h) about 10 ng/mL of EGF;

i) about 5 μg/mL of Transferrin;

j) about 1.5 ng/mL of triiodo-L-thyronine; and

k) about 10 ng/mL of ciprofloxacin hydrochloride,

for a time period sufficient to propagate the one or plurality of primary keratinocytes for no less than about one day. In some embodiments, the exposing step comprising exposing the primary keratinocyte to cell culture media comprising:

a) DMEM/HAM F12(3:1) supplemented with 10% FBS;

b) 1% penicillin/streptomycin;

c) 1% antibiotic-antimycotic;

d) 3 μg/mL of adenine;

e) 3 ng/mL of cholera toxin;

f) 40 ng/mL of hydrocortisone;

g) 5 μg/mL of insulin;

h) 10 ng/mL of EGF;

i) 5 μg/mL of Transferrin;

j) 1.5 ng/mL of triiodo-L-thyronine; and

k) 10 ng/mL of ciprofloxacin hydrochloride,

for a time period sufficient to propagate the one or plurality of primary keratinocytes for no less than about three days. In some embodiments, the exposing step comprising exposing the primary keratinocyte to cell culture media comprising:

a) DMEM/HAM F12(3:1) supplemented with 10% FBS;

b) about 1% penicillin/streptomycin;

c) about 1% antibiotic-antimycotic;

d) about 3 μg/mL of adenine;

e) about 3 ng/mL of cholera toxin;

about 40 ng/mL of hydrocortisone;

g) about 5 μg/mL of insulin;

h) about 10 ng/mL of EGF;

i) about 5 μg/mL of Transferrin;

j) about 1.5 ng/mL of triiodo-L-thyronine; and

k) about 10 ng/mL of ciprofloxacin hydrochloride,

for a time period sufficient to propagate the one or plurality of primary keratinocytes for no less than about seven days. In some embodiments, the step of exposing further comprises positioning a layer of hydrogel underneath the layer of tissue comprising ECM.

In some embodiments, the method of the present disclosure further comprises a step of exposing the one or plurality of primary keratinocytes to one or a plurality of therapeutics. In some embodiments, the therapeutic is a nucleic acid molecule, a peptide or a small molecule, or any combination thereof.

In some embodiments, the method of the present disclosure further comprises monitoring morphology of the one or plurality of primary keratinocytes in response to exposure to the one or plurality of therapeutics.

In some embodiments, the culture system in the method of the present disclosure comprises from about 10 to about 100 inserts.

In another aspect, the present disclosure relates to a method of screening a compound for biological activity comprising exposing one or a plurality of compounds to one or a plurality of genome-edited keratinocytes comprised in any of the compositions disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a representative diagram of intralayers in epithelium tissue.

FIG. 2 shows a model of molecular progression from premalignant lesions to invasive disease.

FIG. 3 shows a schematic of the genome-editing by CRISPR/cas9 transfection followed by FACS sorting based on GFP expression.

FIG. 4A shows a schematic of the pSpCas9(BB)-2A-GFP vector map and FIG. 4B shows a schematic of linearized pSpCas9(BB)-2A-GFP vector used in Example 1.

FIG. 5 shows the percentage of GFP positive cells detected after up to 64 hours post-transfection.

FIG. 6 shows the percentage of primary keratinocytes showing genome-editing 24 hours after transfection.

FIG. 7 shows the genome editing efficiency measured by TIDE before and after FACS selection. TIDE results showing genome-editing efficiency (percentage of cells harboring mutations in the FAT1 gene loci) for non-sorted (top) and sorted (bottom) populations.

FIG. 8 shows the percentage of genome-edited keratinocytes in multiple conditions (in each condition, one single gene was targeted with CRISPR/Cas9) over different passages.

FIG. 9 shows the recovery of genome-edited keratinocytes harboring one single mutation in the genome following FACS-selection.

FIG. 10 shows a schematic representation of a stand design, including the 3D view, top view, side view, and front view.

FIG. 11 shows a ratio of ledge surface and cavity area of a stand from a top view.

FIG. 12 shows the organotypic culture protocol.

FIG. 13 shows the structure of the organotypic cultures of certain genome-edited keratinocytes containing multiple mutations.

FIG. 14A shows the image obtained from squamous cell carcinoma tumor and FIG. 14B shows the structure of the TP53+CDKN2A+NOTCH1 mutated keratinocytes after 2, 3 or 4 weeks of organotypic culture.

FIG. 15A-15F show that single gene mutation is not sufficient for keratinocyte transformation. FIG. 15A: Significantly altered genes with loss of function mutations in head and neck squamous cell carcinoma (TCGA). Columns represent individual tumors; Gray dots, point mutations; light gray bars, gene deletion; dark gray bars, gene amplification. FIG. 15B: Percentage of primary human keratinocytes harboring mutations in specific genes, 24 hours post-transfection (p<0.05 for all individual results). FIG. 15C: Percentage of primary human keratinocytes harboring mutations in a single gene over multiple passages. One representative experiment shown from N=2 biological replicates. FIG. 15D: Organotypic skin culture of genome-edited primary human keratinocytes. FIG. 15E: Total number of cells recovered following FACS-selection of genome-engineered keratinocytes. FIG. 15F: Fraction of primary human tumors harboring gene deletions and mutations in TP53, CDKN2A and NOTCH1 in SCC from distinct tissues indicated (7-13).

FIG. 16 shows the number of driver genomic alterations per HNSCC tumor. Histogram depicting the number of HNSCC — TCGA cases (N=496) with genomic alterations (point mutations, deletions or amplifications) in the genes included in this study. Dotted line represents the trend.

FIG. 17A-17D show genomic alterations in postulated drivers of HNSCC. FIG. 17A: Percentage of cases showing genomic alterations (point mutations and deletions) in specified genes. FIG. 17B and FIG. 17C: Percentage of cases showing genomic alterations (point mutations and deletions) in specified genes in addition to alterations in TP53 or TP53 and CDKN2A, respectively. FIG. 17D: Venn diagram depicting tumors with TP53, CDKN2A and NOTCH1 alterations in HNSCC.

FIG. 18A-18E show that TP53, CDKN2A, and NOTCH1 mutations in combination are sufficient for transformation. FIG. 18A: Percentage of primary human keratinocytes harboring mutations in indicated genes over multiple passages. One representative experiment shown from N=3 biological replicates. FIG. 18B: Total number of cells recovered following FACS-selection and passaging of genome-engineered keratinocytes indicating immortalization. FIG. 18C: Percentage of primary human keratinocytes harboring mutations in specific genes for the expanded conditions (p<0.05 for all individual results). FIG. 18D: Total cell numbers from a time course of genome-edited keratinocytes harboring specific mutations. Data points represents mean±s.e.m, n=3 samples per condition and day. Asterisk represents p value <0.05. FIG. 18E: Sphere-forming assay. Data points represent sphere forming frequency±s.d. (n=6 per group). Asterisk represents p value <0.05.

FIG. 19A-19B show cell cycle analysis of genome-edited keratinocytes. FIG. 19A: Pictures of genome-edited keratinocytes taken after 5 days in 2D culture. FIG. 19B: Cell cycle analysis of genome-edited keratinocytes. Data points represents mean±standard deviation, n=3 samples per condition. Asterisk represents p value <0.05.

FIG. 20A-20D show that TP53, CDKN2A, and NOTCH1 mutations drive keratinocyte invasion. FIG. 20A: H&E staining of organotypic cultures performed with genome-edited keratinocytes. The dotted line indicates the location of the basement membrane. FIG. 20B: Immunohistochemical staining showing K167 and P40 markers on organotypic cultures performed with genome-edited keratinocytes. The dotted line indicates the location of the basement membrane. FIG. 20C: H&E staining of organotypic cultures performed with CTN^(mut) keratinocytes, incubated for 3 weeks. The dotted line depicts the location of the basement membrane. FIG. 20D: H&E staining and P40 immunostaining of tumor section from an NSG mouse injected with CTN^(mut) keratinocytes.

FIG. 21 shows genome-edited foreskin keratinocytes. H&E staining of organotypic cultures performed with CTN^(mut) keratinocytes, incubated for 2-4 weeks. Black arrowheads indicate invasive cells. Gray arrowhead indicates keratin pearl. Dotted line depicts the location of the basement membrane.

FIG. 22A-22D show in vivo genome-edited cells injection. FIG. 22A: Genome-edited keratinocytes (CT^(mut) and CTN^(mut) cells) were injected in the flank of NSG mice. 3.5 months after injection, CTN^(mut) cells formed tumors. Bottom panels shows tumor formed by CTN^(mut) keratinocytes. FIG. 22B: Kaplan-Meier plot showing survival rate of mice injected with CT^(mut) and CTN^(mut) cells. FIG. 22C: Percentage of cells from tumors showing alterations in specific genes. FIG. 22D: H&E and p40 immunohistochemical staining of CTN^(mut) keratinocytes-derived tumor.

FIG. 23 shows UCSF500 copy number analysis of genome-edited keratinocytes. Genome-wide copy number analysis of genome-edited keratinocytes.

FIG. 24 shows genome-edited oral (tonsil) keratinocytes. Organotypic cultures with genome-engineered primary human oral keratinocytes. The dotted line depicts the location of the basement membrane. Arrowheads indicate invasive cells.

FIG. 25A-25C show genetic drivers of transformed-keratinocyte invasion. FIG. 25A: Unsupervised hierarchical clustering analysis of transcriptomic variations across genome-engineered keratinocytes. N=3 replicates per condition, except for CT^(mut) when N=2. FIG. 25B: Alterations in the expression of different cellular processes markers. FIG. 25C: Differentially expressed genes (log 2 fold change) between CT^(mut) and CTN^(mut) primary keratinocytes.

FIG. 26A-26B show RNA-seq analysis of genome-edited keratinocytes. RNA-seq analyses of genome-edited keratinocytes including (FIG. 26A) log 2 fold change in gene expression (FIG. 26B) gene-ontology analyses (performed with PANTHER^(35,36)). Left, CDKN2A^(mut) keratinocytes; center, CT^(mut) keratinocytes; and right, CTN^(mut) keratinocytes.

FIG. 27 shows a model of squamous cell carcinoma formation and progression.

FIG. 28A-28B shows prevalence of mutations in TP53, CDKN2A and NOTCH1 in normal skin. FIG. 28A: Number of biopsies showing non-synonymous alterations in TP53, CDKN2A and NOTCH1^(17,18). FIG. 28B: Variant allele fraction (VAF) for each mutation in the 4 biopsies from esophageal tissue study¹⁸ that show presence of mutations in TP53, CDKN2A and NOTCH1.

DETAILED DESCRIPTION OF EMBODIMENTS

The present disclosure can be understood more readily by reference to the following detailed description of embodiments, the figures and the examples included herein.

Before the present methods and compositions are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

Moreover, it is to be understood that unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, and the number or type of aspects described in the specification.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

As used in the specification and in the claims, the term “comprising” can include the aspects “consisting of” and “consisting essentially of” Comprising can also mean “including but not limited to.”

As used in the specification and the appended claims, the singular forms “a,” “an” and “the” can include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes mixtures of compounds; reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

The word “or” as used herein means any one member of a particular list and also includes any combination of members of that list.

The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. According to certain embodiments, when referring to a measurable value such as an amount and the like, “about” is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2% or ±0.1% from the specified value as such variations are appropriate to perform the disclosed methods. When “about” is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

As used herein, the term “primary” cell type means that the cells are derived directly from tissue comprising the named cells. In some embodiments, the cells may be human or non-human animal cells. In some embodiments, the cells are primary human, non-human primate, dog, pig, horse, cow, sheep, goat, cat, mouse, rat cells. In some embodiments, the primary cells are derived from a human, non-human primate, dog, pig, horse, cow, goat, sheep, cat, mouse, rat or other non-human animal.

As used herein, the term “deficient in expression” encompasses deficient in protein function (partial or complete loss of function), with altered function as compared to wild-type expression, or with gain of function. In some embodiments, the cells deficient in expression carrying the identified gene do not express a sufficient number of wild-type amino acid sequences to confer a wild-type biological function. In some embodiments, the cells deficient in expression are free of one or more of the identified genes. In some embodiments, the cells deficient in expression are free of one or more of the identified wild-type genes. In some embodiments, the cells deficient in expression are free of a portion or the full coding region of one or more of the wild-type TP53, CDKN2A and NOTCH1. In some embodiments, the cells deficient in expression express only a non-biologically functional or mutated form of the peptide encoded by the identified nucleic acid sequence. In some embodiments, the cells deficient in expression express one or more amino acid sequences encoded by a nucleic acid comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to one or a combination of: SEQ ID NO: 311, SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315 or 316, provided that the nucleic acid does not comprise the nucleic acid sequence of SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO:309.

The “percent identity” or “percent homology” of two polynucleotide or two polypeptide sequences is determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. “Identical” or “identity” as used herein in the context of two or more nucleic acids or amino acid sequences, may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may he performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0. Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length Win the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached. The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, which is incorporated herein by reference in its entirety) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873-5787, which is incorporated herein by reference in its entirety) and Gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance. For example, a nucleic acid is considered similar to another if the smallest sum probability in comparison of the test nucleic acid to the other nucleic acid is less than about 1, less than about 0.1, less than about 0.01, and less than about 0.001.

As used herein, the terms “genetically altering,” “genome-edited,” or “genome editing” refers to genetic alternation(s) of one or a plurality of wild-type genes in animal and plant cells, including human cells, which may be knock-out or knock-in of a specific gene, or introducing a mutation into a non-coding DNA sequence that does not produce protein. The term “genome-edited” may include deletion, duplication, inversion, replacement or rearrangement of genomic DNA. In some embodiments, only a single wild-type gene is genetically altered or edited in the cell. In some embodiments, more than one, such as 2, 3, 4, 5, or more regions of one gene or a plurality of genes are altered or edited in the cell. Techniques for genome editing or introducing a targeted mutation into the nucleotide sequence of a gene are known in the art, including but not limited to the use of mutagens.

As used herein, the term “mutagen” means any energenic wave, any molecule, nucleic acid sequence, amino acid sequence, or hybrid amino acid or nucleic acid sequence that causes a mutation or modification in one or more regions of endogenous nucleic acid when exposed for a time period sufficient to cause the mutation. In some embodiments, the mutation is a point mutation, frameshift mutation, deletion, truncation, or addition. In some embodiments, the mutagen is a vector or a gene-modifying enzyme.

The term “vector” as used herein refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. Vectors can be delivered into cells ex vivo by any methods known in the art, including but not limited to, non-viral transfection, biolistics, chemical transfection, electroporation, nucleofection, heat-shock transfection, lipofection, microinjection, and viral transfection.

The terms “transfection,” “transduction,” “transfecting” or “transducing” can be used interchangeably and are defined as a process of introducing a nucleic acid molecule and/or a protein to a cell. Nucleic acids may be introduced to a cell using viral-based or non-viral methods. The nucleic acid molecule can be a sequence encoding complete proteins or functional portions thereof. Typically, a nucleic acid vector, comprising the elements necessary for protein expression (e.g., a promoter, transcription start site, etc.), is used. For viral-based methods, any useful viral vector can be used in the methods described herein. Examples of viral vectors include, but not limited to, retroviral, adenoviral, lentiviral and adeno-associated viral vectors. In some embodiments, the nucleic acid molecules are introduced into a cell using a retroviral vector following standard procedures well known in the art. Non-viral methods of transfection include any appropriate method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell. Exemplary non-viral transfection methods include, but not limited to, calcium phosphate transfection (or chemical transfection), liposomal transfection (or lipofection), nucleofection, sonoporation, transfection through heat shock (or heat-shock transfection), microinjection, biolistics, magnetifection and electroporation. In some embodiments, cell transfection is carried out by electroporation. In some embodiments, cell transfection is carried out by nucleofection. In some embodiments, cell transfection is carried out by lipofection. The terms “transfection” or “transduction” also refer to introducing ribonucleoprotein complexs (or ribonucleoprotein particles or RNPs) or proteins into a cell from the external environment. Typically, transduction or transfection of a protein relies on attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, e.g., Ford et al. (2001) Gene Therapy 8:1-4 and Prochiantz (2007) Nat. Methods 4:119-20.

The term “electroporation,” as used herein, refers to application of an electrical current or electrical field to a cell to facilitate entry of a nucleic acid molecule into the cell. During this process, the electrical current or electrical field applied to the cell increases the permeability of the cell membrane, allowing chemicals, drugs, or nucleic acids (e.g., DNA, RNA, or DNA/RNA hybrid) to be introduced into the cell (also called electrotransfer).

A “ribonucleoprotein complex,” or “ribonucleoprotein particle” or “RNP” as used herein refers to a complex or particle including a nucleoprotein and a ribonucleic acid. A “nucleoprotein” as provided herein refers to a protein capable of binding a nucleic acid (e.g., RNA, DNA). Where the nucleoprotein binds a ribonucleic acid, it is referred to as “ribonucleoprotein.” The interaction between the ribonucleoprotein and the ribonucleic acid may be direct, e.g., by covalent bond, or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like). In some embodiments, the ribonucleoprotein includes an RNA-binding motif non-covalently bound to the ribonucleic acid. For example, positively charged aromatic amino acid residues (e.g., lysine residues) in the RNA-binding motif may form electrostatic interactions with the negative nucleic acid phosphate backbones of the RNA, thereby forming a ribonucleoprotein complex. Non-limiting examples of ribonucleoproteins include ribosomes, telomerase, RNAseP, hnRNP, CRISPR associated protein 9 (Cas9) and small nuclear RNPs (snRNPs). The ribonucleoprotein may be an enzyme. In some embodiments, the ribonucleoprotein is an endonuclease. In some embodiments, the ribonucleoprotein complex includes an endonuclease and a ribonucleic acid. In some embodiments, the endonuclease is a CRISPR associated protein 9 (Cas9). In some embodiments therefore, the RNPs used in the methods of the disclosure are CRISPR/Cas9 ribonucleoprotein (RNP) complexes.

The term “gene-modifying enzyme” as used herein refers to an enzyme that is capable of modifying a gene by introducing a mutation (e.g., point mutation, frameshift mutation, deletion, or truncation) causing gene inactivation or introducing heterologous nucleotides (e.g., genes) through non-homologous end joining or homologous recombination. Exemplary gene-modifying enzymes, include but not limited to, a Cas protein, a meganuclease, a transcription activator-like effector nucleases (TALEN), a transposon, a zinc-finger nuclease (ZFN), or a recombinase. In some embodiments, the gene-modifying enzyme suitable for the methods disclosed herein is a Cas protein, a meganuclease, a TALEN, a ZFN, or a recombinase. In some embodiments, the gene-modifying enzyme suitable for the methods disclosed herein is a Cas protein. In some preferred embodiments, the gene-modifying enzyme suitable for the methods disclosed herein is a Cas9 protein.

The term “Cas9 protein” refers to the “clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein 9.” This term is well known in the art and has been described, e.g. in Makarova et al. (2011) Nat. Rev. Microbiol., 9:467-477, and in Makarova et al. (2011) Biol. Direct., 6:38.

Cas proteins are endonuclease that form part of an adaptive defense mechanism evolved by bacteria and archaea to protect them from invading viruses and plasmids. Cas9 protein or gene information can be obtained from a known database such as the GenBank of NCBI (National Center for Biotechnology Information), but is not limited thereto. Moreover, the Cas9 protein may comprise not only wild-type Cas9, but also deactivated Cas9 (dCas9), or Cas9 variants such as Cas9 nickase. The deactivated Cas9 may be RFN (RNA-guided FokI nuclease) comprising a FokI nuclease domain bound to dCas9, or may be dCas9 to which a transcription activator or repressor domain is bound. In addition, the Cas9 protein is not limited in its origin. For example, the Cas9 protein may be derived from Streptococcus pyogenes, Francisella novicida, Streptococcus thermophiles, Legionella pneumophila, Listeria innocua, or Streptococcus mutans.

Cas9 protein is the major protein element of the CRISPR/Cas9 system, which forms a complex with crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) to form activated endonuclease or nickase. “CRISPR system” refers collectively to transcripts or synthetically produced transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). In the context of formation of a CRISPR complex, “target sequence” refers to a nucleic acid sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, the target sequence is a DNA polynucleotide and is referred to a DNA target sequence. In some embodiments, a target sequence comprises at least three nucleic acid sequences that are recognized by a Cas-protein when the Cas protein is associated with a CRISPR complex or system which comprises at least one sgRNA or one tracrRNA/crRNA duplex at a concentration and within an microenvironment suitable for association of such a system. In some embodiments, the target DNA comprises at least one or more proto-spacer adjacent motifs which sequences are known in the art and are dependent upon the Cas protein system being used in conjunction with the sgRNA or crRNA/tracrRNAs employed by this work. In some embodiments, the target DNA comprises NNG, where G is a guanine and N is any naturally occurring nucleic acid. In some embodiments the target DNA comprises any one or combination of NNG, NNA, GAA, NNAGAAW and NGGNG, where G is a guanine, A is adenine, and N is any naturally occurring nucleic acid.

Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. without wishing to be bound by theory, the tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. In some embodiments, the tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of a CRISPR complex. As with the target sequence, it is believed that complete complementarity is not needed, provided there is sufficient to be functional (bind the Cas protein or functional fragment thereof). In some embodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell such that the presence and/or expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector.

With at least some of the modification contemplated by this disclosure, in some embodiments, the guide sequence or RNA or DNA sequences that form a CRISPR complex are at least partially synthetic. The CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5^(mut) with respect to (“upstream” of) or 3′ with respect to (“downstream” of) a second element. In some embodiments, the disclosure relates to a composition comprising a chemically synthesized guide sequence. In some embodiments, the chemically synthesized guide sequence is used in conjunction with a vector comprising a coding sequence that encodes a CRISPR enzyme, such as a type II Cas9 protein. In some embodiments, the chemically synthesized guide sequence is used in conjunction with one or more vectors, wherein each vector comprises a coding sequence that encodes a CRISPR enzyme, such as a type II Cas9 protein. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more additional (second, third, fourth, etc.) guide sequences, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, one or more additional guide sequence, tracr mate sequence, and tracr sequence are each a component of different nucleic acid sequences. For instance, in the case of a tracr and tracr mate sequences and in some embodiments, the disclosure relates to a composition comprising at least a first and second nucleic acid sequence, wherein the first nucleic acid sequence comprises a tracr sequence and the second nucleic acid sequence comprises a tracr mate sequence, wherein the first nucleic acid sequence is at least partially complementary to the second nucleic acid sequence such that the first and second nucleic acid for a duplex and wherein the first nucleic acid and the second nucleic acid either individually or collectively comprise a DNA-targeting domain, a Cas protein binding domain, and a transcription terminator domain. In some embodiments, the CRISPR enzyme, one or more additional guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter. In some embodiments, the disclosure relates to compositions comprising any one or combination of the disclosed domains on one guide sequence or two separate tracrRNA/crRNA sequences with or without any of the disclosed modifications. Any methods disclosed herein also relate to the use of tracrRNA/crRNA sequence interchangeably with the use of a guide sequence, such that a composition may comprise a single synthetic guide sequence and/or a synthetic tracrRNA/crRNA with any one or combination of modified domains disclosed herein.

The CRISPR system suitable for the present disclosure can also comprise a modified CRISPR enzyme (or “Cas protein”) or a nucleotide sequence encoding one or more Cas proteins. Any protein capable of enzymatic activity in cooperation with a guide sequence is a Cas protein. In some embodiments, the disclosure relates to a system comprises a vector comprising a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein from the Cas family of enzymes. In some embodiments, the disclosure relates to a system, composition, or pharmaceutical composition comprising any one or plurality of Cas proteins either individually or in combination with one or a plurality of guide sequences. Compositions of one or a plurality of Cas proteins may be administered to a subject with any of the disclosed guide sequences sequentially or contemporaneously. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, type V CRISPR-Cas systems, variants and fragments thereof, or modified versions thereof having at least 70% identity to the sequences of Table X. These enzymes are known, for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In some embodiments the CRISPR enzyme is Cas9, and may be Cas9 from S. pyogenes or S. pneumoniae. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In some embodiments, a vector encodes a CRISPR enzyme or Cas protein that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A. In some embodiments, a Cas9 nickase may be used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ.

As a further example, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III) may be mutated to produce a mutated Cas9 substantially lacking all DNA cleavage activity. In some embodiments, a D10A mutation is combined with one or more of H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially lacking all DNA cleavage activity. In some embodiments, a CRISPR enzyme is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or lower with respect to its non-mutated form. Other mutations may be useful; where the Cas9 or other CRISPR enzyme is from a species other than S. pyogenes, mutations in corresponding amino acids may be made to achieve similar effects.

TABLE X Accession numbers of Cas proteins (or those related with Cas-like function) and nucleic acids encoding the same. All amino acid and nucleic acid sequences associated with the Accession Numbers below are incorporated by reference in their entireties. 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Meganucleases are by definition sequence-specific endonucleases with large (>14 bp) cleavage sites that can deliver DNA double-strand breaks at specific loci in living cells (Thierry and Dujon, Nucleic Acids Res., 1992, 20, 5625-5631). Meganucleases have been used to stimulate homologous recombination in the vicinity of their target sequences in cultured cells and plants (Rouet et al., Mol. Cell. Biol., 1994, 14, 8096-106; Choulika et al., Mol. Cell. Biol., 1995, 15, 1968-73; Donoho et al., Mol. Cell. Biol, 1998, 18, 4070-8; Elliott et al., Mol. Cell. Biol., 1998, 18, 93-101; Sargent et al., Mol. Cell. Biol., 1997, 17, 267-77; Puchta et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 5055-60; Chiurazzi et al., Plant Cell, 1996, 8, 2057-2066), making meganuclease-induced recombination an efficient and robust method for genome engineering.

In nature, meganucleases are essentially represented by homing endonucleases (HEs), a family of endonucleases encoded by mobile genetic elements, whose function is to initiate DNA double-strand break-induced recombination events in a process referred to as homing (Chevalier and Stoddard, Nucleic Acids Res., 2001, 29, 3757-74; Kostriken et al., Cell; 1983, 35, 167-74; Jacquier and Dujon, Cell, 1985, 41, 383-94). Several hundreds of HEs have been identified in bacteria, eukaryotes, and archea (Chevalier and Stoddard, Nucleic Acids Res., 2001, 29, 3757-74). In addition to those found in nature, meganucleases suitable for practicing the methods of the present disclosure may also be functional variants or functional fragments of wild-type meganucleases.

Transcription activator-like effector nucleases, or “TALEN,” are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations (Boch, J., Nature Biotechnology, 29 (2):135-6).

A transposon, or a transposable element (TE) or jumping gene, is a DNA sequence that can change its position within a genome, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size (Bourque et al. (2018) Genome Biology, 19 (1): 199). There are at least two classes of transposons, Class I transposons or retrotransposons generally function via reverse transcription and Class II transposons or DNA transposons encode the protein transposase, which they require for insertion and excision, and some of these transposons also encode other proteins (Pray, L. A. (2008) Nature Education, 1(1): 204). Transposons suitable for practicing the methods of the present disclosure may comprise not only wild-type transposons, but also functional fragments thereof.

Zinc-finger nucleases, or ZFNs, are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. The modular DNA recognition preferences of zinc-fingers protein allows for the rational design of site-specific multi-finger DNA binding proteins. Fusion of the nuclease domain from the Type II restriction enzyme Fok I to site-specific zinc-finger proteins allows for the creation of site-specific nucleases. See, e.g., US 2003/0232410; US 2005/0208489; US 2005/0026157; US 2005/0064474; US 2006/0188987; US 2006/0063231; US 2007/0134796; US 2008/015164; US 2008/0131962; US 2008/015996; WO 07/014275; and WO 2008/133938, which all describe use of zinc-finger nucleases.

Recombinases are genetic recombination enzymes. Exemplary recombinases include, but not limited to, site-specific recombinases which are derived from bacteria and fungi and catalyze directionally sensitive DNA exchange reactions between short (30-40 nucleotides) target site sequences that are specific to each recombinase. These reactions enable excision/insertion, inversion, translocation, and cassette exchange (Nern et al. (2011) PNAS, 108(34):14198-203; Garcia-Otin et al. (2006) Frontiers in Bioscience, 11:1108-36; Dymecki et al. (2007) Neuron, 54(1):17-34; Luan et al. (2007) Current Opinion in Neurobiology, 17(5):572-80; Fenno et al. (2014) Nature Methods, 11(7):763-72). Recombinases suitable for practicing the methods of the present disclosure may comprise not only wild-type recombinases, but also functional fragments thereof.

A “functional fragment” of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains some or all of the function as the full-length protein, polypeptide or nucleic acid. A functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well-known in the art. Similarly, methods for determining protein function are well-known. For example, the DNA-binding function of a polypeptide can be determined by, for instance, filter-binding, electrophoretic mobility-shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis. See e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates. The ability of a protein to interact with another protein can be determined by, for example, co-immunoprecipitation, two-hybrid assays or complementation, both genetic and biochemical. See e.g., Fields et al. (1989) Nature 340:245-246; U.S. Pat. No. 5,585,245 and WO 98/44350.

The term “plasmid” refers to a DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. Thus, a plasmid is considered a replicon or a unit of DNA capable of replicating autonomously within a suitable host.

The term “hydrogel” refers to a network of polymer chains that are hydrophilic, sometimes found as a colloidal gel in which water is the dispersion medium. In some embodiments, the hydrogel suitable for practicing the present disclosure is Matrigel, which is a trade name for a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells produced and marketed by Corning Life Sciences and BD Biosciences. The main components of Matrigel® are structural proteins such as laminin, nidogen, collagen and heparan sulfate proteoglycans which present cultured cells with the adhesive peptide sequences that they would encounter in their natural environment (Hughes et al. (2010) Proteomics, 10(9):1886-90). Also present are growth factors like TGF-β and EGF that prevent differentiation and promote proliferation of many cell types.

As used herein, the terms “squamous cell carcinoma” or “SCC” means a cancer of a kind of epithelial cell, the squamous cell that make up the main component or part of the epidermis of the skin.

SCC is one of the major forms of skin cancer. However, squamous cells also occur in the lining of the digestive tract, lungs, and other areas of the body, and SCC occurs as a form of cancer in diverse tissues, including the lips, mouth, esophagus, urinary bladder, prostate, lung, vagina, and cervix, among others. SCC is a histologically distinct form of cancer arising from the uncontrolled multiplication of cells of epithelium, or cells showing particular cytological or tissue architectural characteristics of squamous cell differentiation, such as the presence of keratin, tonofilament bundles, or desmosomes, structures involved in cell-to-cell adhesion. SCC is sometimes referred to as “epidermoid carcinoma” and “squamous cell epithelioma.”

Genome-Edited Keratinocytes

The disclosure relates to a composition comprising one or a plurality of genome-edited keratinocytes. In some embodiment, the keratinocyte is a primary cell from a human or mammal. Genome-edited, or genetically altered, keratinocytes can be prepared by exposing primary keratinocytes to one or a plurality of mutagens for a time period sufficient to mutate one or a plurality of wild-type genes such that genome-edited primary keratinocytes deficient in expression of the one or plurality of wild-type genes are produced. Mutagens suitable for causing genetic mutation(s) can be any type of physical or chemical mutagens known in the art, such as radioactive substances, radiation, and chemical substances. Mutagens can also be molecules that frequently used in laboratories to introduce genetic mutation(s), such as vectors or gene-modifying enzymes. In some embodiments, the mutagen is a gene-modifying enzyme, such as a Cas protein, a meganuclease, a transcription activator-like effector nucleases (TALEN), a transposon, a zinc-finger nuclease (ZFN), or a recombinase, but not limited thereto. In some embodiments, the mutagen is a vector, such as a plasmid, but not limited thereto.

In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of one or a plurality of wild-type genes, particularity those provided in Table 1.

TABLE 1 List of target genes for genome editing. Gene Genomic Sequence Target Transcript TP53 NC_000017.11 NM_000546.5 NOTCH1 NC_000009.12 NM_017617.3 CDKN2A NC_000009.12 NM_001195132.1 TP63 NC_000003.12 NM_001114980.1 FAT1 NC_000004.12 NM_005245.3 CASP8 NC_000002.12 NM_001080125.1 RB1 NC_000013.11 NM_000321.2 PTEN NC_000010.11 NM_001304718.1 NSD1 NC_000005.10 NM_022455.4 KMT2D NC_000012.12 NM_003482.3 AJUBA NC_000014.9 NM_032876.5 EP300 NC_000022.11 NM_001429.3 EPHA2 NC_000001.11 NM_004431.3 FBXW7 NC_000004.12 NM_033632.3 RASA1 NC_000005.10 NM_002890.2 TGFBR2 NC_000003.12 NM_003242.5 ZNF750 NC_000017.11 NM_024702.2 KEAP1 NC_000019.10 NM_203500.1 HRAS NC_000011.10 NM_001130442.1 PIK3CA NC_000003.12 NM_006218.2 RHOA NC_000003.12 NM_001664.2 CCR5 NC_000003.12 NM_000579.3 AAVS1 NC_000019.10 NM_017607.4

In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of a single wild-type gene. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of a single wild-type gene chosen from Table 1. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type TP53 gene. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild-type CDKN2A gene. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild-type NOTCH1 gene.

In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of at least two wild-type genes. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of at least one wild-type gene chosen from Table 1 and at least one further wild-type gene. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type TP53 gene and at least one further wild-type gene. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type CDKN2A gene and at least one further wild-type gene. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type NOTCH1 gene and at least one further wild-type gene. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of at least two wild-type genes chosen from Table 1. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type TP53 and CDKN2A genes. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type TP53 and NOTCH1 genes. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type CDKN2A and NOTCH1 genes.

In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of at least three wild-type genes. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of at least one wild-type gene chosen from Table 1 and at least two further wild-type gene. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type TP53 gene and two further wild-type genes. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type CDKN2A gene and two further wild-type genes. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type NOTCH1 gene and two further wild-type genes. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of at least two wild-type genes chosen from Table 1 and at least one further wild-type gene. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type TP53 and CDKN2A genes and one further wild-type gene. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type TP53 and NOTCH1 genes and one further wild-type gene. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type CDKN2A and NOTCH1 genes and one further wild-type gene. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of at least three wild-type genes chosen from Table 1. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type TP53, CDKN2A and NOTCH1 genes.

In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of four or more wild-type genes. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of at least one wild-type gene chosen from Table 1 and at least three or more wild-type genes. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type TP53 gene and three or more wild type genes. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type CDKN2A gene and three or more wild type genes. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type NOTCH1 gene and three or more wild type genes. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of at least two wild-type genes chosen from Table 1 and at least tow or more wild-type genes. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type TP53 and CDKN2A genes and two or more wild type genes. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type TP53 and NOTCH1 genes and two or more wild type genes. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type CDKN2A and NOTCH1 genes and two or more wild type genes. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of at least three wild-type gene chosen from Table 1 and at least one or more wild-type genes. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of wild type TP53, CDKN2A and NOTCH1 genes and one or more wild type genes. In some embodiments, the genome-edited keratinocytes according to the present disclosure are deficient in expression of four or more wild-type genes chosen from Table 1.

In some embodiments, the genome-edited keratinocytes according to the present disclosure comprises at least one mutated gene having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 311 or 312, provided that it does not comprise the nucleic acid sequence of SEQ ID NO: 307. In some embodiments, the genome-edited keratinocytes according to the present disclosure comprises at least one mutated gene comprising the nucleic acid sequence of SEQ ID NO: 311 or 312. In some embodiments, the genome-edited keratinocytes according to the present disclosure comprises at least one mutated gene having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 313 or 314, provided that it does not comprise the nucleic acid sequence of SEQ ID NO: 308. In some embodiments, the genome-edited keratinocytes according to the present disclosure comprises at least one mutated gene comprising the nucleic acid sequence of SEQ ID NO: 313 or 314. In some embodiments, the genome-edited keratinocytes according to the present disclosure comprises at least one mutated gene having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 315 or 316, provided that it is free of the nucleic acid sequence of SEQ ID NO: 309. In some embodiments, the genome-edited keratinocytes according to the present disclosure comprises at least one mutated gene comprising the nucleic acid sequence of SEQ ID NO: 315 or 316.

In some embodiments, the genome-edited keratinocytes according to the present disclosure can be prepared by transfecting primary keratinocytes with a plasmid, cosmid or nucleic acid sequence complexed with protein. Primary keratinocytes can be isolated from skin tissue or mucosa, such as oral mucosa, mucosa of the upper aerodigestive tract, esophageal mucosa, vaginal mucosa, or cervical mucosa, using any method known in the art or those disclosed herein. Transfection of primary keratinocytes can be performed using any method known in the art. In some embodiments, the transfection of primary keratinocytes are performed by seeding primary keratinocytes on tissue culture-treated polystyrene multi-well plates at a density of about 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000 or more cells per well or per milliliter of volume, followed by incubation with plasmid for transfection for about 2, 4, 6, 8, 12, 24, 36, 48 or more hours.

In some embodiments, the primary keratinocytes are transfected with one or a plurality of plasmids or vectors. In some embodiments, the primary keratinocytes are transfected with one or a plurality of ribonucleoprotein (RNP) complexes. In some embodiments, the primary keratinocytes are transfected with one or a plurality of CRISPR/Cas9 ribonucleoprotein (RNP) complexes. In some embodiments, the transfection of primary keratinocytes are carried out by electroporation. This can be accomplished using electroporation devices that are configured to deliver to primary keratinocytes pulse of energy effective to cause reversible pores to form in cell membranes, and preferable the pulse of energy is a constant current similar to a preset current input by a user. The electroporation device may comprise an electroporation component and an electrode assembly or handle assembly. The electroporation component may include and incorporate one or more of the various elements of the electroporation devices, including: controller, current waveform generator, impedance tester, waveform logger, input element, status reporting element, communication port, memory component, power source, and power switch.

The amount of nucleic acid (e.g., ssDNA, dsDNA, RNA) that may be introduced into the primary keratinocytes can be from about 0.5 nanograms to about 50 micrograms. However, this amount may be varied to optimize transfection efficiency and/or cell viability. In some embodiments, less than about 100 picograms of nucleic acid may be added to each cell sample (e.g., one or more cells being electroporated). In some embodiments, at least about 100 picograms, at least about 200 picograms, at least about 300 picograms, at least about 400 picograms, at least about 500 picograms, at least about 600 picograms, at least about 700 picograms, at least about 800 picograms, at least about 900 picograms, at least about 1 microgram, at least about 1.5 micrograms, at least about 2 micrograms, at least about 2.5 micrograms, at least about 3 micrograms, at least about 3.5 micrograms, at least about 4 micrograms, at least about 4.5 micrograms, at least about 5 micrograms, at least about 5.5 micrograms, at least about 6 micrograms, at least about 6.5 micrograms, at least about 7 micrograms, at least about 7.5 micrograms, at least about 8 micrograms, at least about 8.5 micrograms, at least about 9 micrograms, at least about 9.5 micrograms, at least about 10 micrograms, at least about 11 micrograms, at least about 12 micrograms, at least about 13 micrograms, at least about 14 micrograms, at least about 15 micrograms, at least about 20 micrograms, at least about 25 micrograms, at least about 30 micrograms, at least about 35 micrograms, at least about 40 micrograms, at least about 45 micrograms, or at least about 50 micrograms, of nucleic acid may be added to each cell sample (e.g., one or more cells being electroporated).

Electroporation using, for example, the Neon® Transfection System (ThermoFisher Scientific), the AMAXA® Nucleofector (AMAXA Biosystems), or the MaxCyte ATx instrument (MAXCYTE, Inc.) can also be used for delivery of nucleic acids into primary keratinocytes. Electroporation parameters may be adjusted to optimize transfection efficiency and/or cell viability. Electroporation devices can have multiple electrical wave form pulse settings such as exponential decay, time constant and square wave. Every cell type has a unique optimal Field Strength (E) that is dependent on the pulse parameters applied (e.g., voltage, capacitance and resistance). Application of optimal field strength causes electropermeabilization through induction of transmembrane voltage, which allows nucleic acids to pass through the cell membrane. In some embodiments, the electroporation pulse voltage, the electroporation pulse width, number of pulses, cell density, and tip type may be adjusted to optimize transfection efficiency and/or cell viability.

In some embodiments, electroporation pulse voltage may be varied to optimize transfection efficiency and/or cell viability. In some embodiments, the electroporation voltage may be less than about 500 volts. In some embodiments, the electroporation voltage may be at least about 500 volts, at least about 600 volts, at least about 700 volts, at least about 800 volts, at least about 900 volts, at least about 1000 volts, at least about 1100 volts, at least about 1200 volts, at least about 1300 volts, at least about 1400 volts, at least about 1500 volts, at least about 1600 volts, at least about 1700 volts, at least about 1800 volts, at least about 1900 volts, at least about 2000 volts, at least about 2100 volts, at least about 2200 volts, at least about 2300 volts, at least about 2400 volts, at least about 2500 volts, at least about 2600 volts, at least about 2700 volts, at least about 2800 volts, at least about 2900 volts, or at least about 3000 volts.

In some embodiments, electroporation pulse width may be varied to optimize transfection efficiency and/or cell viability. In some embodiments, any of the methods disclosed herein comprise the electroporation pulse width may be less than about 5 milliseconds. In some embodiments, the electroporation width may be at least about 5 milliseconds, at least about 6 milliseconds, at least about 7 milliseconds, at least about 8 milliseconds, at least about 9 milliseconds, at least about 10 milliseconds, at least about 11 milliseconds, at least about 12 milliseconds, at least about 13 milliseconds, at least about 14 milliseconds, at least about 15 milliseconds, at least about 16 milliseconds, at least about 17 milliseconds, at least about 18 milliseconds, at least about 19 milliseconds, at least about 20 milliseconds, at least about 21 milliseconds, at least about 22 milliseconds, at least about 23 milliseconds, at least about 24 milliseconds, at least about 25 milliseconds, at least about 26 milliseconds, at least about 27 milliseconds, at least about 28 milliseconds, at least about 29 milliseconds, at least about 30 milliseconds, at least about 31 milliseconds, at least about 32 milliseconds, at least about 33 milliseconds, at least about 34 milliseconds, at least about 35 milliseconds, at least about 36 milliseconds, at least about 37 milliseconds, at least about 38 milliseconds, at least about 39 milliseconds, at least about 40 milliseconds, at least about 41 milliseconds, at least about 42 milliseconds, at least about 43 milliseconds, at least about 44 milliseconds, at least about 45 milliseconds, at least about 46 milliseconds, at least about 47 milliseconds, at least about 48 milliseconds, at least about 49 milliseconds, or at least about 50 milliseconds.

In some embodiments, the number of electroporation pulses may be varied to optimize transfection efficiency and/or cell viability. In some embodiments, electroporation may comprise a single pulse. In some embodiments, electroporation may comprise more than one pulse. In some embodiments, electroporation may comprise 2 pulses, 3 pulses, 4 pulses, 5 pulses 6 pulses, 7 pulses, 8 pulses, 9 pulses, or 10 or more pulses.

In some embodiments, the starting cell density for electroporation may be varied to optimize transfection efficiency and/or cell viability. In some embodiments, the starting cell density for electroporation may be less than about 1×10⁵ cells. In some embodiments, the starting cell density for electroporation may be at least about 1×10⁵ cells, at least about 2×10⁵ cells, at least about 3×10⁵ cells, at least about 4×10⁵ cells, at least about 5×10⁵ cells, at least about 6×10⁵ cells, at least about 7×10⁵ cells, at least about 8×10⁵ cells, at least about 9×10⁵ cells, at least about 1×10⁶ cells, at least about 1.5×10⁶ cells, at least about 2×10⁶ cells, at least about 2.5×10⁶ cells, at least about 3×10⁶ cells, at least about 3.5×10⁶ cells, at least about 4×10⁶ cells, at least about 4.5×10⁶ cells, at least about 5×10⁶ cells, at least about 5.5×10⁶ cells, at least about 6×10⁶ cells, at least about 6.5×10⁶ cells, at least about 7×10⁶ cells, at least about 7.5×10⁶ cells, at least about 8×10⁶ cells, at least about 8.5×10⁶ cells, at least about 9×10⁶ cells, at least about 9.5×10⁶ cells, at least about 1×10⁷ cells, at least about 1.2×10⁷ cells, at least about 1.4×10⁷ cells, at least about 1.6×10⁷ cells, at least about 1.8×10⁷ cells, at least about 2×10⁷ cells, at least about 2.2×10⁷ cells, at least about 2.4×10⁷ cells, at least about 2.6×10⁷ cells, at least about 2.8×10⁷ cells, at least about 3×10⁷ cells, at least about 3.2×10⁷ cells, at least about 3.4×10⁷ cells, at least about 3.6×10⁷ cells, at least about 3.8×10⁷ cells, at least about 4×10⁷ cells, at least about 4.2×10⁷ cells, at least about 4.4×10⁷ cells, at least about 4.6×10⁷ cells, at least about 4.8×10⁷ cells, or at least about 5×10⁷ cells.

Additional exemplary nucleic acid delivery systems include those provided by AMAXA® Biosystems (Cologne, Germany), Life Technologies (Frederick, Md.), MAXCYTE, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) and Copernicus Therapeutics Inc. (see for example U.S. Pat. No. 6,008,336).

The transfection efficiency of primary keratinocytes with any of the nucleic acid delivery platforms described herein, for example, electroporation or lipofection, can be about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more than 99.9%. The cell viability and/or transfection efficiency may be measured using any method known in the art, including but not limited to, trypan blue exclusion, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), the presence or absence of given cell-surface markers (e.g., CD4 or CD8), telomere length, fluorescence-activated cell sorting (FACS), real-time PCR, or droplet digital PCR. In some embodiments, FACS may be used to detect the cell viability and/or transfection efficiency. In other embodiments, apoptosis of cells may be measured using a TUNEL assay.

In some embodiments, transfected primary keratinocytes can be selected and separated from non-transfected primary keratinocytes. However, selection and separation of transfected primary keratinocytes is not an essential step of the methods disclosed herein. In some embodiments therefore, the methods of the disclosure do not include a step of selecting and/or sorting transfected primary keratinocytes. When this selection step is used, transfected keratinocytes can be selected by FACS in some embodiments. In some embodiments, transfected keratinocytes can be sorted onto tissue culture-treated polystyrene multi-well plates.

Depending on the transfection efficiency and whether transfected primary keratinocytes are selected and sorted, a population of genome-edited primary keratinocytes according to the disclosure may comprise non-transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises from about 50% to about 100% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises from about 45% to about 95% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises from about 40% to about 90% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises from about 35% to about 85% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises from about 30% to about 80% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises from about 25% to about 75% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises from about 20% to about 70% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises from about 15% to about 65% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises from about 10% to about 60% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises about 100% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises about 95% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises about 90% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises about 85% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises about 80% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises about 75% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises about 70% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises about 65% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises about 60% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises about 55% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises about 50% of transfected primary keratinocytes. In some embodiments, a population of genome-edited primary keratinocytes according to the disclosure comprises about 45%, 40%, 35%, 30%, 25%, 20% 15%, 10%, 5% or less than about 5% of transfected primary keratinocytes.

In some embodiments, the plasmid for transfection comprises a nucleic acid sequence encoding a gene-modifying enzyme. Any gene-modifying enzyme known in the art can be used. Examples of gene-modifying enzymes include, but not limited to Cas proteins, meganucleases, transcription activator-like effector nucleases (TALENs), transposons, zinc-finger nucleases (ZFNs), or recombinases, or functional fragments thereof. A functional fragment of such a gene-modifying enzyme may comprise an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more sequence identity to the parent gene-modifying enzyme. Representatives of such gene-modifying enzymes are provided in Table 2. Functional fragments of these representative gene-modifying enzymes therefore may comprise an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

TABLE 2 Representatives of gene-modifying enzymes. SEQ ID Name Sequence NO Cas9 MDKKYSIGLDIGTNSVGWAVITDEY 16 KVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRR KNRICYLQEIFSNEMAKVDDSFFHR LEESFLVEEDKKHERHPIFGNIVDE VAYHEKYPTIYHLRKKLVDSTDKAD LRLIYLALAHMIKFRGHFLIEGDLN PDNSDVDKLFIQLVQTYNQLFEENP INASGVDAKAILSARLSKSRRLENL IAQLPGEKKNGLFGNLIALSLGLTP NFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRY DEHHQDLTLLKALVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKF IKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRR QEDFYPFLKDNREKIEKILTFRIPY YVGPLARGNSRFAWMTRKSEETITP WNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELT KVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECF DSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTL FEDREMIEERLKTYAHLFDDKVMKQ LKRRRYTGWGRLSRKLINGIRDKQS GKTILDFLKSDGFANRNFMQLIHDD SLTFKEDIQKAQVSGQGDSLHEHIA NLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQK NSRERMKRIEEGIKELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVD QELDINRLSDYDVDHIVPQSFLKDD SIDNKVLTRSDKNRGKSDNVPSEEV VKKMKNYWRQLLNAKLITQRKFDNL TKAERGGLSELDKAGFIKRQLVETR QITKHVAQILDSRMNTKYDENDKLI REVKVITLKSKLVSDFRKDFQFYKV REINNYHHAHDAYLNAVVGTALIKK YPKLESEFVYGDYKVYDVRKMIAKS EQEIGKATAKYFFYSNIMNFFKTEI TLANGEIRKRPLIETNGETGEIVWD KGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDKLIARKKD WDPKKYGGFDSPTVAYSVLVVAKVE KGKSKKLKSVKELLGITIMERSSFE KNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPE DNEQKQLFVEQHKHYLDEIIEQISE FSKRVILADANLDKVLSAYNKHRDK PIREQAENIIHLFTLTNLGAPAAFK YFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGDSRADPKK KRKV Cas9_ST MDKKYSIGLDIGTNSVGWAVITDEY 17 RP1 KVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRR KNRICYLQEIFSNEMAKVDDSFFHR LEESFLVEEDKKHERHPIFGNIVDE VAYHEKYPTIYHLRKKLVDSTDKAD LRLIYLALAHMIKFRGHFLIEGDLN PDNSDVDKLFIQLVQTYNQLFEENP INASGVDAKAILSARLSKSRRLENL IAQLPGEKKNGLFGNLIALSLGLTP NFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRY DEHHQDLTLLKALVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKF IKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRR QEDFYPFLKDNREKIEKILTFRIPY YVGPLARGNSRFAWMTRKSEETITP WNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELT KVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECF DSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTL FEDREMIEERLKTYAHLFDDKVMKQ LKRRRYTGWGRLSRKLINGIRDKQS GKTILDFLKSDGFANRNFMQLIHDD SLTFKEDIQKAQVSGQGDSLHEHIA NLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQK NSRERMKRIEEGIKELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVD QELDINRLSDYDVDHIVPQSFLKDD SIDNKVLTRSDKNRGKSDNVPSEEV VKKMKNYWRQLLNAKLITQRKFDNL TKAERGGLSELDKAGFIKRQLVETR QITKHVAQILDSRMNTKYDENDKLI REVKVITLKSKLVSDFRKDFQFYKV REINNYHHAHDAYLNAVVGTALIKK YPKLESEFVYGDYKVYDVRKMIAKS EQEIGKATAKYFFYSNIMNFFKTEI TLANGEIRKRPLIETNGETGEIVWD KGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDKLIARKKD WDPKKYGGFDSPTVAYSVLVVAKVE KGKSKKLKSVKELLGITIMERSSFE KNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPE DNEQKQLFVEQHKHYLDEIIEQISE FSKRVILADANLDKVLSAYNKHRDK PIREQAENIIHLFTLTNLGAPAAFK YFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD Cas3 MKHINDYFWAKKTEENSRLLWLPLT 18 QHLEDTKNIAGLLWEHWLSEGQKVL IENSINVKSNIENQGKRLAQFLGAV HDIGKATPAFQTQKGYANSVDLDIQ LLEKLERAGFSGISSLQLASPKKSH HSIAGQYLLSHYGVDEDIATIIGGH HGRPVDDLDGLNSQKSYPSNYYQDE KKDSLVYQKWKSNQEAFLNWALTET GFNSVSQLPKIKQPAQVILSGLLIM SDWIASNEHFFPLLSLDETDVKNKS QRIETGFKKWKKSNLWQPETFVDLV TLYQERFGFSPRNFQLILSQTIEKT TNPGFVILEAPMGIGKTEAALAVSE QLSSKKGCSGLFFGLPTQATSNGIF KRIEQWTENIKGNNSDHFSIQLVHG KAALNTDFIELLKGNTINMDDSENG SIFVNEWFSGRKTSALDDFVVGTVD QFLMVALKQKHLALRHLGFSKKVIV IDEVHAYDAYMSQYLLEAIRWMGAY GVPVIILSATLPAQQREKLIKSYMA GMGVKWRDIENIDQIKIDAYPLITY NDGPDIHQVKMFEKQEQKNIYIHRL PEEQLFDIVKEGLDNGGVVGIIVNT VRKSQELARNFSDIFGDDMVDLLHS NFIATERIRKEKDLLQEIGKKAIRP PKKIIIGTQVLEQSLDIDFDVLISD LAPMDLLIQRIGRLHRHKIKRPQKH EVARFYVLGTFEEFDFDEGTRLVYG DYLLARTQYFLPDKIRLPDDISPLV QKVYNSDLTITFPKPELHKKYLDAK IEHDDKIKNKETKAKSYRIANPVLK KSRVRTNSLIGWLKNLHPNDSEEKA YAQVRDIEDTVEVIALKKISDGYGL FIENKDISQNITDPIIAKKVAQNTL RLPMSLSKAYNIDQTINELERYNNS HLSQWQNSSWLKGSLGIIFDKNNEF ILNGFKLLYDEKYGVTIERLDKNES V Cas4 MITEFLLKKKLEEHLSHVKEENTIY 19 VTDLVRCPRRVRYESEYKELAISQV YAPSAILGDILHLGLESVLKGNFNA ETEVETLREINVGGKVYKIKGRADA IIRNDNGKSFVIEIKTSRSDKGLPL IHHKMQLQIYLWLFSAEKGILVYIT PDRIAEYEINEPLDEATIVRLAEDT IMLQNSPRFNWECKYCIFSVICPAK Cas5 MSPHIDANGVPIDCLSFTVRSTWGH 20 FKRVGRSVTKQTYRIPPRTTVAGML AAFVGADRDSYYDVFGADNSAIAIT PLSDLRTVNIPTTGLGTDPKQDVTE TAGSWRTYKLTYQNTTGDRQLHSYE VLADPAYRIDVALENEEFYDSLREH LVNGTSIYPPSLGKSEYLAVIEDVE VDREPEVQDIDETLDIDSVVPISLS EAVPQGKVTYGVERSPAVMVRESGG RRTTRFDDYVYAQQASNPVRIGEQT DVAPVQVGDRTVVFR PIGA L1 MGPKKKRKVAAADYKDDDDKSRPGE 21 RPFQCRICMRNFSDSPTLRRHTRTH TGEKPFQCRICMRNFSQRSSLVRHL RTHTGEKPFQCRICMRNFSERGNLT RHLKTHLRGSQLVKSELEEKKSELR HKLKYVPHEYIELIEIARNSTQDRI LEMKVMEFFMKVYGYRGKHLGGSRK PDGAIYTVGSPIDYGVIVDTKAYSG GYNLPIGQADEMQRYVEENQTRNKH INPNEWWKVYPSSVTEFKFLFVSGH FKGNYKAQLTRLNHITNCNGAVLSV EELLIGGEMIKAGTLTLEEVRRKFN NGEINF PIGA R1 MGPKKKRKVAAADYKDDDDKSRPGE 22 RPFQCRICMRNFSAPSKLKRHTRTH TGEKPFQCRICMRNFSHKSSLTRHL RTHTGEKPFQCRICMRNFSQRNALA GHLKTHLRGSQLVKSELEEKKSELR HKLKYVPHEYIELIEIARNSTQDRI LEMKVMEFFMKVYGYRGKHLGGSRK PDGAIYTVGSPIDYGVIVDTKAYSG GYNLPIGQADEMQRYVEENQTRNKH INPNEWWKVYPSSVTEFKFLFVSGH FKGNYKAQLTRLNHITNCNGAVLSV EELLIGGEMIKAGTLTLEEVRRKFN NGEINF AAVS1 MAPKKKRKVGIHGVPAAMAERPFQC 23 R1 RICMRNFSQSSNLARHIRTHTGEKP FACDICGRKFARTDYLVDHTKIHTG SQKPFQCRICMRNFSYNTHLTRHIR THTGEKPFACDICGRKFAQGYNLAG HTKIHLRGSQLVKSELEEKKSELRH KLKYVPHEYIELIEIARNSTQDRIL EMKVMEFFMKVYGYRGKHLGGSRKP DGAIYTVGSPIDYGVFVDTKAYSGG YNLPIGQADEMQRYVKENQTRNKHI NPNEWWKVYPSSVTEFKFLFVSGHF KGNYKAQLTRLNHKTNCNGAVLSVE ELLIGGEMIKAGTLTLEEVRRKFNN GEINF* AAVS1 MAPKKKRKVGIHGVPAAMAERPFQC 24 L1 RICMRNFSYNWHLQRHIRTHTGEKP FACDICGRKFARSDHLTTHTKIHTG SQKPFQCRICMRNFSHNYARDCHIR THTGEKPFACDICGRKFAQNSTRIG HTKIHLRGSQLVKSELEEKKSELRH KLKYVPHEYIELIEIARNSTQDRIL EMKVMEFFMKVYGYRGKHLGGSRKP DGAIYTVGSPIDYGVFVDTKAYSGG YNLPIGQADEMERYVEENQTRNKHL NPNEWWKVYPSSVTEFKFLFVSGHF KGNYKAQLTRLNHITNCNGAVLSVE ELLIGGEMIKAGTLTLEEVRRKFNN GEINF* phiC31 MDTYAGAYDRQSRERENSSAASPAT 25 Integrase QRSANEDKAADLQREVERDGGRFRF VGHFSEAPGTSAFGTAERPEFERIL NECRAGRLNMIIVYDVSRFSRLKVM DAIPFVSELLALGVTIVSTQEGVFR QGNVMDLIHLIMRLDASHKESSLKS AKILDTKNLQRELGGYVGGKAPYGF ELVSETKEITRNGRMVNVVINKLAH STTPLTGPFEFEPDVIRWWWREIKT HKHLPFKPGSQAAIHPGSITGLCKR MDADAVPTRGETIGKKTASSAWDPA TVMRILRDPRIAGFAAEVIYKKKPD GTPTTKIEGYRIQRDPITLRPVELD CGPIIEPAEWYELQAWLDGRGRGKG LSRGQAILSAMDKLYCECGAVMTSK RGEESIKDSYRCRRRKVVDPSAPGQ HEGTCNVSMAALDKFVAERIFNKIR HAEGDEETLALLWEAARRFGKLTEA PEKSGERANLVAERADALNALEELY EDRAAGAYDGPVGRKHFRKQQAALT LRQQGAEERLAELEAAEAPKLPLDQ WFPEDADADPTGPKSWWGRASVDDK RVFVGLFVDKTVVTKSTTGRGQGTP IEKRASITWAKPPTDDDEDDAQDGT EDVAA I-CreI MNTKYNKEFLLYLAGFVDGDGSIIA 26 QIKPNQSYKFKHQLSLTFQVTQKTQ RRWFLDKLVDEIGVGYVRDRGSVSD YILSEIKPLHNFLTQLQPFLKLKQK QANLVLKIIEQLPSAKESPDKFLEV CTWVDQIAALNDSKTRKTTSETVRA VLDSLSEKKKSSP I-SceI MKNIKKNQVMNLGPNSKLLKEYKSQ 27 LIELNIEQFEAGIGLILGDAYIRSR DEGKTYCMQFEWKNKAYMDHVCLLY DQWVLSPPHKKERVNHLGNLVITWG AQTFKHQAFNKLANLFIVNNKKTIP NNLVENYLTPMSLAYWFMDDGGKWD YNKNSTNKSFVLNTQSFTFEEVEYL VKGLRNKFQLNCYVKINKNKPIIYI DSMSYLIFYNLIKPYLIPQMMYKLP NTISSETFLK RAG1 MAASFPPTLGLSSAPDEIQHPHIKF 28 SEWKFKLFRVRSFEKTPEEAQKEKK DSFEGKPSLEQSPAVLDKADGQKPV PTQPLLKAHPKFSKKFHDNEKARGK AIHQANLRHLCRICGNSFRADEHNR RYPVHGPVDGKTLGLLRKKEKRATS WPDLIAKVFRIDVKADVDSIHPTEF CHNCWSIMHRKFSSAPCEVYFPRNV TMEWHPHTPSCDICNTARRGLKRKS LQPNLQLSKKLKTVLDQARQARQHK RRAQARISSKDVMKKIANCSKIHLS TKLLAVDFPEHFVKSISCQICEHIL ADPVETNCKHVFCRVCILRCLKVMG SYCPSCRYPCFPTDLESPVKSFLSV LNSLMVKCPAKECNEEVSLEKYNHH ISSHKESKEIFVHINKGGRPRQHLL SLTRRAQKHRLRELKLQVKAFADKE EGGDVKSVCMTLFLLALRARNEHRQ ADELEAIMQGKGSGLQPAVCLAIRV NTFLSCSQYHKMYRTVKAITGRQIF QPLHALRNAEKVLLPGYHHFEWQPP LKNVSSSTDVGIIDGLSGLSSSVDD YPVDTIAKRFRYDSALVSALMDMEE DILEGMRSQDLDDYLNGPFTVVVKE SCDGMGDVSEKHGSGPVVPEKAVRF SFTIMKITIAHSSQNVKVFEEAKPN SELCCKPLCLMLADESDHETLTAIL SPLIAEREAMKSSELMLELGGILRT FKFIFRGTGYDEKLVREVEGLEASG SVYICTLCDATRLEASQNLVFHSIT RSHAENLERYEVWRSNPYHESVEEL RDRVKGVSAKPFIETVPSIDALHCD IGNAAEFYKIFQLEIGEVYKNPNAS KEERKRWQATLDKHLRKKMNLKPIM RMNGNFARKLMTKETVDAVCELIPS EERHEALRELMDLYLKMKPVWRSSC PAKECPESLCQYSFNSQRFAELLST KFKYRYEGKITNYFHKTLAHVPEII ERDGSIGAWASEGNESGNKLFRRFR KMNARQSKCYEMEDVLKHHWLYTSK YLQKFMNAHNALKTSGFTMNPQASL GDPLGIEDSLESQDSMEF Sleeping MGKSKEISQDLRKRIVDLHKSGSSL 29 Beauty GAISKRLAVPRSSVQTIVRKYKHHG Transposon TTQPSYRSGRRRVLSPRDERTLVRK (SB100X) VQINPRTTAKDLVKMLEETGTKVSI STVKRVLYRHNLKGHSARKKPLLQN RHKKARLRFATAHGDKDRTFWRNVL WSDETKIELFGHNDHRYVWRKKGEA CKPKNTIPTVKHGGGSIMLWGCFAA GGTGALHKID TALEN MSRTRLPSPPAPSPAFSADSFSDLL 30 RQFDPSLFNTSLFDSLPPFGAHHTE AATGEWDEVQSGLRAADAPPPTMRV AVTAARPPRAKPAPRRRAAQPSDAS PAAQVDLRTLGYSQQQQEKIKPKVR STVAQHHEALVGHGFTHAHFVALSQ HPAALGTVAVKYQDMIAALPEATHE AIVGVGKQWSGARALEALLTVAGEL RGPPLQLDTGQLLKIAKRGGVTAVE AVHAWRNALTGAPLNLTETVHETHG TASQLTPEQVVAIASNIGGRPALES IVAQLSRPDPALAALTNDHLVALAC LGGRPALDAVKKGLPHAPALIKRTN RRIPERTSHRVADHAQVVRVLGFFQ CHSHPAQAFDDAMTQFGMSRHGLLQ LFRRVGVTELEARSGTLPPASQRWD RILQASGMKRAKPSPTSTQTPDQAS LHAFADSLERDLDAPSPMHEGDQTR ASASPKKKRKVEASGSGRADALDDF DLDMLGSDALDDFDLDMLGSDALDD FDLDMLGSDALDDFDLDMLIN

In some embodiments, the plasmid for transfection comprises a nucleic acid sequence encoding a Cas9 protein. Cas9 is an RNA-guided DNA endonuclease that cleaves a DNA substrate having sequence complementary to the guide RNA. The guide RNA has a constant part and a variable part. The variable part contains 20 nucleotides, the sequence of which will guide the Cas9 protein to the specific site in the genome to create a double strand break. Thus, in some embodiments, the plasmid for transfection further comprises a guide RNA comprising a variable part having a nucleic acid sequence that is complementary to a portion of a target gene to be modified. In some embodiments, more than one 20-nucleotide variable parts, such as 2, 3, 4, 5, or more, are used for a single target gene.

In some embodiments, the plasmid for transfection further comprises a marker gene to facilitate the selection of transfected keratinocytes. Any marker gene known in the art can be used. Examples of marker genes include, but not limited to, fluorescent reporter genes, antibiotic resistance genes and surface protein-coding genes. Functional fragments of the proteins encoded by such marker genes, for instance, functional fragments having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the parent protein can also be used. Proteins encoded by representative marker genes are provided in Table 3. Functional fragments of these proteins encoded by the representative marker genes therefore may comprise an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320, SEQ ID NO: 321, SEQ ID NO: 322, SEQ ID NO: 323.

TABLE 3 Representatives of marker genes. SEQ Name Sequence ID NO GFP VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGK 317 LPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMA DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSA LSKDPNEKRDHMVLLEFVTAAGITLGMDELYK T dtomato MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTK 318 GGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFED GGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLY PRDGVLKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDIT SHNEDYTIVEQYERSEGRHHLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFM RFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSP QFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGLVTVTQDSSL QDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQ ALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQY ERSEGRHHLFLYGMDELYK EBFP2 MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTT 319 GKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFK DDGTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYI MAVKQKNGIKVNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLST QSVLSKDPNEKRDHMVLLEFRTAAGITLGMDELYK mKate2 MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKAVEGG 320 PLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVL TATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGG LEGRAD MALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIK EADKETYVEQHEVAVARYCDLPSKLGH mKO MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVTMAK 321 GGPMPFAFDLVSHVFCYGHRPFTKYPEEIPDYFKQAFPEGLSWERSLEFEDG GSASVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASD GVLKGDVTMYLKLEGGGNHKCQFKTTYKAAKKILKMPGSHYISHRLVRKT EGNITELVEDAVAHS Puromycin MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVTELQE 322 LFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGS RLAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAA ERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA DeltaLNG MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREPGSGAIAKEACPT 323 FR + SBP GLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKP (Streptavidin CTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLV binding FSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWAD peptide) AECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGS SQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR

In some embodiments, the marker gene comprised in the plasmid for transfection encodes a fluorescent reporter of any color, such as green, red, blue or yellow. In some embodiments, the marker gene encodes a green fluorescent protein (GFP). Keratinocytes transfected with a plasmid containing the GFP marker gene generally show GFP fluorescence levels within few hours, such as about 6, 10, 12, 16, 20, 24 or more hours. GFP fluorescence intensity, or other type of fluorescence intensity, can be determined and measured by any methods known in the art. In some embodiments, transfected keratinocytes are selected by Fluorescence-Activated Cell Sorting (FACS). The transfected keratinocytes can be sorted onto tissue culture-treated polystyrene multi-well plates and expanded. Expansion of primary keratinocytes, such as primary human keratinocytes, can be done using any methods known in the art. Thus, in some embodiments, the present disclosure also relates to a method of generating a population of one or a plurality of genome-edited keratinocytes comprising:

-   -   a) generating and selecting one or a plurality of genome-edited         primary keratinocytes deficient in expression of one or a         plurality of wild-type genes according to any method disclosed         herein; and     -   b) expanding the one or plurality of genome-edited primary         keratinocytes into the population of one or plurality of         genome-edited keratinocytes.         A schematic of this genome-editing by CRISPR/cas9 transfection         followed by FACS selection and expansion according to the         present disclosure is shown in FIG. 3 .

This step of selecting transfected keratinocytes, however, is notessential to the methods disclosed herein. In some embodiments therefore, the disclosure also relates to a method of generating a population of one or a plurality of genome-edited keratinocytes comprising:

-   -   a) generating one or a plurality of genome-edited primary         keratinocytes deficient in expression of one or a plurality of         wild-type genes according to any method disclosed herein; and     -   b) expanding the one or plurality of genome-edited primary         keratinocytes into the population of one or plurality of         genome-edited keratinocytes.

Genome-editing in transfected keratinocytes can be checked by any methods known in the art. One example of such methods is TIDE for Tracking of Indels by Decomposition (Brinkman et al., Easy quantitative assessment of genome editing by sequence trace decomposition, Nucleic Acids Res., 2014 Dec., 16; 42(22):e168; accessible at deskgen.com/landing/tide.html#/tide). The TIDE method, which requires only a pair of PCR reactions and two standard capillary sequencing runs, can deconvolute Sanger capillary sequencing reactions to infer the proportion of a population of cells whose genome has been edited. In some embodiments, any method according to the present disclosure for preparing genome-edited keratinocytes can further comprise a step of checking genome editing by using, for example, the TIDE method.

Compositions and Systems

The disclosure relates to a composition comprising one or a plurality of keratinocytes comprising at least three deficient or mutated genes that regulate cell growth. In some embodiments, the mutated genes are deficient in expression of NOTCH1, CDKN2A and TP53. In some embodiments, the cells are primary cells from a mammal. In some embodiments, the cells are primary human cells. In some embodiments, the cells comprise at least one point mutation in each of the NOTCH1, CDKN2A and TP53 genes. In some embodiments, the cell comprise genome-edited mutations in only the NOTCH1, CDKN2A and TP53 genes. In some embodiments, the mutated genes are deficient in expression of NOTCH1, CDKN2A, TP53, and at least one other gene identified in Table 1.

The genome-edited keratinocytes according to the present disclosure can be used in a composition, which can comprise one or more additional components. In some embodiments, such compositions include a solid support or culture dish comprising a well. In some embodiments, the well further comprise a top layer of cells and a bottom layer of tissue, the bottom layer of tissue comprising extracellular matrix protein and the top layer of cells comprising one or a plurality of genome-edited keratinocytes or those cells from the aforementioned paragraph according to the present disclosure. The disclosure relates to a cell culture system comprising a solid support comprising at least one vessel or insert, Any tissue comprising extracellular matrix protein, including but not limited to dermis and cadaver skin, can be used. In some embodiments, the bottom layer of tissue comprises one or a plurality of fibroblasts. In some embodiments, the compositions of the present disclosure further comprise a hydrogel layer positioned underneath the bottom layer of tissue. In some embodiments, the hydrogel layer comprises one or a plurality of growth factors or functional fragments thereof. Suitable growth factors include, but not limited to the growth factors provided in Table 4.

TABLE 4 List of growth factors. Growth SEQ Factor Sequence ID NO EGF MLLTLIILLPVVSKFSFVSLSAPQHWSCPEGTLAGNGNSTCVGPAPFLIFSHGNS 340 IFRIDTEGTNYEQLVVDAGVSVIMDFHYNEKRIYWVDLERQLLQRVFLNGSRQ ERVCNIEKNVSGMAINWINEEVIWSNQQEGIITVTDMKGNNSHILLSALKYPA NVAVDPVERFIFWSSEVAGSLYRADLDGVGVKALLETSEKITAVSLDVLDKRL FWIQYNREGSNSLICSCDYDGGSVHISKHPTQHNLFAMSLFGDRIFYSTWKMK TIWIANKHTGKDMVRINLHSSFVPLGELKVVHPLAQPKAEDDTWEPEQKLCK LRKGNCSSTVCGQDLQSHLCMCAEGYALSRDRKYCEDVNECAFWNHGCTLG CKNTPGSYYCTCPVGFVLLPDGKRCHQLVSCPRNVSECSHDCVLTSEGPLCFC PEGSVLERDGKTCSGCSSPDNGGCSQLCVPLSPVSWECDCFPGYDLQLDEKSC AASGPQPFLLFANSQDIRHMHFDGTDYGTLLSQQMGMVYALDHDPVENKIYF AHTALKWIERANMDGSQRERLIEEGVDVPEGLAVDWIGRRFYWTDRGKSLIG RSDLNGKRSKIITKENISQPRGIAVHPMAKRLFWTDTGINPRIESSSLQGLGRLV IASSDLIWPSGITIDFLTDKLYWCDAKQSVIEMANLDGSKRRRLTQNDVGHPF AVAVFEDYVWFSDWAMPSVMRVNKRTGKDRVRLQGSMLKPSSLVVVHPLA KPGADPCLYQNGGCEHICKKRLGTAWCSCREGFMKASDGKTCLALDGHQLL AGGEVDLKNQVTPLDILSKTRVSEDNITESQHMLVAEIMVSDQDDCAPVGCS MYARCISEGEDATCQCLKGFAGDGKLCSDIDECEMGVPVCPPASSKCINTEGG YVCRCSEGYQGDGIHCLDIDECQLGEHSCGENASCTNTEGGYTCMCAGRLSE PGLICPDSTPPPHLREDDHHYSVRNSDSECPLSHDGYCLHDGVCMYIEALDKY ACNCWGYIGERCQYRDLKWWELRHAGHGQQQKVIWAVCVWLVMLLLL SLWGAHYYRTQKLLSKNPKNPYEESSRDVRSRRPADTEDGMSSCPQPWFVVI KEHQDLKNGGQPVAGEDGQAADGSMQPTSWRQEPQLCGMGTEQGCWIPVSS DKGSCPQVMERSFHMPSYGTQTLEGGVEKPHSLLSANPLWQQRALDPPHQME LTQ bFGF MVGVGGGDVEDVTPRPGGCQISGRGARGCNGIPGAAAWEAALPRRRPRRHPS 341 VNPRSRAAGSPRTRGRRTEERPSGSRLGDRGRGRALPGGRLGGRGRGRAPER VGGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTLPALPEDGGSGAFPP GHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKG VCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVAL KRTGQYKLGSKTGPGQKAILFLPMSAKS NGF MSMLFYTLITAFLIGIQAEPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSA 342 PAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEV GGAAPFNRTHRSKRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVL GEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALT MDGKQAAWRFIRIDTACVCVLSRKAVRRA PDGF MNRCWALFLSLCCYLRLVSAEGDPIPEELYEMLSDHSIRSFDDLQRLLHGDPG 343 EEDGAELDLNMTRSHSGGELESLARGRRSLGSLTIAEPAMIAECKTRTEVFEIS RRLIDRTNANFLVWPPCVEVQRCSGCCNNRNVQCRPTQVQLRPVQVRKIEIVR KKPIFKKATVTLEDHLACKCETVAAARPVTRSPGGSQEQRAKTPQTRVTIRTV RVRRPPKGKHRKFKHTHDKTALKETLGA IGF-1 MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAGPETLC 344 GAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGFVDECCFRSCDLRRLEM YCAPLKPAKSARSVRAQRHTDMPKTQKYQPPSTNKNTKSQRRKGWPKTHPG GEQKEGTEASLQIRGKKKEQRREIGSRNAECRGKKGK TGF-β MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILS 345 KLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEV TRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKL KVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEI EGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLER AQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHA NFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGR KPKVEQLSNMIVRSCKCS

In some embodiments, the hydrogel layer comprises one or a plurality of growth factors chosen from Table 4 or a functional fragment thereof comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the growth factors listed in Table 4. The hydrogel used in the compositions of the present disclosure can be those that are commercially available, such as Matrigel® and Cultrex®. Matrigel® is the trade name for a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells produced and marketed by Corning Life Sciences and BD Biosciences. The base components of Matrigel® are 60% laminin, 30% collagen IV, and 8% entactin. In some embodiments, the Matrigel further comprises the following growth factors:

Growth Factor Concentration EGF From about 0.5 to about 1.3 ng/mL bFGF From about 0.01-0.2 pg/mL NGF From about 0.01 ng/mL to about 0.2 ng/mL PDGF From about 5 pg mL to about 48 pg/mL IGF-1 From about 11 ng/mL to about 24 ng/mL TGF-β From about 1.7 ng/mL to about 4.7 ng/mL Cultrex® is the trade name for a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumor and marketed by Trevigen, Inc. In some embodiments, the hydrogel used in the compositions of the present disclosure is Matrigel®. In some embodiments, the compositions of the present disclosure is at about 30° C., 35° C., or 37° C. and exposed to about 3%, 4% or 5% CO2 for no less than about 24 or 48 hours.

In some embodiments, the compositions of the present disclosure further comprise at least a single insert, (e.g. portable plastic well with a flat surface whose surface area is defined by one or a plurality of sidewalls, the insert or well comprising a biocompatible polymer and an inner wall defining an inner volume divided into an upper chamber and a bottom chamber, the upper chamber and bottom chamber positioned adjacently to one another and in fluid communication and separated by a ledge; the ledge positioned along an inner wall of the insert and defining a planar interface between the upper and bottom chambers. In some embodiments, the compositions of the present disclosure comprise a top layer of cells, a bottom layer of tissue and a hydrogel; each of the top layer of cells and bottom layer of tissue positioned in the upper chamber; and wherein the hydrogel is positioned in the bottom chamber; wherein the bottom layer of tissue comprises extracellular matrix protein and the top layer of cells comprises one or a plurality of genome-edited keratinocytes keratinocytes according to the present disclosure. The compositions of the present disclosure can comprise more than one insert, such as 2, 6, 12, 24, 48, 96, 192 or more inserts. Thus, in some embodiments, the compositions of the present disclosure relates to a solid support comprising at least about 2, 6, 12, 24, 48, 96, 192 or more inserts. In some embodiments, the inserts are equally sized and positioned in a pattern at discrete positions on or within the solid support.

In some embodiments, the interface of the compositions of the present disclosure is a planar or substantially planar opening with a surface area from about 0.01 to about 20, about 0.05 to about 10, from about 0.1 to about 5, about 0.2 to about 3 square centimeters. In some embodiments, the upper chamber of the compositions of the present disclosure has a volume from about 0.5 to about 20, about 0.3 to about 15, about 0.2 to about 10, about 0.1 to about 9, about 0.05 to about 5 centimeters cubed. In some embodiments, the bottom chamber of the compositions of the present disclosure has a volume from about 0.5 to about 20, about 0.3 to about 15, about 0.2 to about 10, about 0.1 to about 9, about 0.05 to about 5 centimeters cubed. In some embodiments, the compositions of the present disclosure are frozen at from about −20° C. to about −212° C.

The genome-edited keratinocytes according to the present disclosure can be used in a system comprising:

-   -   a) at least one solid support comprising a bottom surface and         sidewalls, the bottom surface and sidewalls defining a culture         volume;     -   b) at least one insert as described herein positioned on the         bottom surface of the at least one solid support; and     -   c) one or a plurality of genome-edited keratinocytes according         to the present disclosure.

In some embodiments, the systems of the present disclosure further comprise a top layer of cells and a bottom layer of tissue, the bottom layer of tissue comprising extracellular matrix protein and the top layer of cells comprising the one or plurality of keratinocytes. In some embodiments, the bottom layer of tissue comprises one or a plurality of fibroblasts.

In some embodiments, the systems of the present disclosure further comprise a hydrogel layer as described herein positioned adjacently underneath the bottom layer of tissue. In some embodiments, the hydrogel is Matrigel®.

In some embodiments, the systems of the present disclosure further comprise an incubator in which the solid support is positioned and wherein the incubator comprises a heating element and an air inlet through which air content is controlled. The incubator maintains the temperature of the systems at about 30° C., 35° C., or 37° C. and comprises ambient air in contact with the solid support that comprises about 3%, 4% or 5% CO2. The systems of the present disclosure can further comprise culture media in the volume of the solid support with a cell culture media volume sufficient to cover the solid support and expose one or plurality of keratinocytes contained therein to air. In some embodiments, the system of the present disclosure further comprises a tissue culture medium at a volume sufficient to cover the bottom tissue and create an air liquid interface immediately adjacent to or below the top layer of cells, such that the top layer of cells is exposed to air. In some embodiments, the air is filtered or sterilized in a closed system.

The solid support of the systems of the present disclosure can be from about 1 to about 10, about 2 to about 8, or about 3 to about 5 centimeters in height.

In some embodiments, the systems of the present disclosure comprise a top layer of cells, a bottom layer of tissue and a hydrogel; each of the top layer of cells and bottom layer of tissue positioned in the upper chamber; and wherein the hydrogel positioned in the bottom chamber; wherein the bottom layer of tissue comprises extracellular matrix protein and the top layer of cells comprises the one or plurality of primary keratinocytes. The systems of the present disclosure can comprise more than one insert, such as 2, 6, 12, 24, 48, 96, 192 or more inserts. Thus, in some embodiments, the systems of the present disclosure comprise at least about 2, 6, 12, 24, 48, 96, 192 or more inserts.

In some embodiments, the interface of the systems of the present disclosure is a planar or substantially planar opening with a surface area from about 0.01 to about 20, about 0.05 to about 10, from about 0.1 to about 5, about 0.2 to about 3 square centimeters. In some embodiments, the upper chamber of the systems of the present disclosure has a volume from about 0.5 to about 20, about 0.3 to about 15, about 0.2 to about 10, about 0.1 to about 9, about 0.05 to about 5 centimeters cubed. In some embodiments, the bottom chamber of the systems of the present disclosure has a volume from about 0.5 to about 20, about 0.3 to about 15, about 0.2 to about 10, about 0.1 to about 9, about 0.05 to about 5 centimeters cubed. In some embodiments, the systems of the present disclosure further comprise a tissue culture medium at a volume sufficient to cover the top layer of cells.

The systems of the present disclosure can further comprise a gas reservoir in fluid communication with the solid support via one or a plurality of gas lines; and a heating element operably positioned at or proximate to the solid support, such that the temperature of ambient air around the top layer of cells is from about 32° C. to about 42°.

The systems of the present disclosure can further comprise one or more therapeutic compounds. The therapeutic can be a nucleic acid molecule, a peptide or a small molecule, or any combination thereof

Methods

In one aspect, the present disclosure related to a method of isolating a primary keratinocytes from skin tissue or mucosa comprising culturing a primary keratinocyte to culture medium while simultaneously exposing one or more keratinocytes to one or a plurality of tissue samples comprising extracellular matrix (ECM). The skin tissue can be obtained from any part of a human body, such as forehead, face, neck, head, or leg, but not limited thereto. The skin tissue can also be anal skin. The mucosa can be mucosa of any source, such as oral mucosa, mucosa of the upper aerodigestive tract, esophageal mucosa, vaginal mucosa, or cervical mucosa, but not limited thereto.

The present disclosure further relates to a method of culturing a primary keratinocyte comprising exposing the primary keratinocyte to a system comprising:

-   -   a) at least one solid support comprising a bottom surface and         sidewalls, the bottom surface and sidewalls defining a culture         volume; and     -   b) at least one insert as described herein positioned on the         bottom surface of the at least one solid support; wherein the         insert comprises a layer of tissue comprising ECM onto which the         primary keratinocytes are positioned.

The step of exposing can further comprise exposing the primary keratinocyte to cell culture media comprising:

a) DMEM/HAM F12(3:1) supplemented with 10% FBS;

b) 1% penicillin/streptomycin;

c) 1% antibiotic-antimycotic;

d) 3 μg/mL of adenine;

e) 3 ng/mL of cholera toxin;

f) 40 ng/mL of hydrocortisone;

g) 5 μg/mL of insulin;

h) 10 ng/mL of EGF;

i) 5 μg/mL of Transferrin;

j) 1.5 ng/mL of triiodo-L-thyronine; and

k) 10 ng/mL of ciprofloxacin hydrochloride,

for a time period sufficient to propagate the one or plurality of primary keratinocytes for no less than about one, two, three, four, five, six, seven, eight, nine or ten days. The step of exposing can further comprise positioning a layer of hydrogel as described herein underneath the layer of tissue comprising ECM. In some embodiments the time period sufficient to propagate the one or plurality of primary keratinocytes further comprises exposing th cells to about 5% oxygen in air and culturing the cells at about 37 degrees Celsius.

In some embodiments, the disclosure relates to a method of manufacturing a system or culture of primary keratinocytes using any one or more method steps disclosed herein and further comprising fixing the tissue in culture. In some embodiments, fixation can be achieved by exposing the tissue to ethanol or formalin. In some embodiments, cultures may be embedded in paraffin or embedded in OCT.

The method can further comprise a step of exposing the one or plurality of primary keratinocytes to one or a plurality of therapeutics or agents. The therapeutic can be a nucleic acid molecule, a peptide or a small molecule, or any combination thereof. A step of monitoring morphology of the one or plurality of primary keratinocytes in response to exposure to the one or plurality of therapeutics can also be included in the method.

The disclosure relates to a method of altering the genetic material of a primary keratinocyte comprising exposing the primary keratinocyte to one or a plurality of gene modifying enzymes, such as a Cas9 protein, that are specific for NOTCH1, CDKN2A and TP53. In some embodiments, the cells are primary cells from a mammal. In some embodiments, the cells are primary human cells. In some embodiments, the cells comprise at least one point mutation in each of the NOTCH1, CDKN2A and TP53 genes. In some embodiments, the exposing step results in the cells comprising genome-edited mutations in only the NOTCH1, CDKN2A and TP53 genes. In some embodiments, the mutated genes are deficient in expression of NOTCH1, CDKN2A, TP53, and at least one other gene identified in Table 1. In some embodiments the method of altering the primary keratinocyte comprises the step of isolating the one or plurality of keratinocytes prior to the exposure step. In some embodiments, the method of altering the primary keratinocyte comprises the step of culturing the primary keratinocytes with any of the conditions or duration of time disclosed herein.

The culture systems used in such methods can comprise from about 1 to about 200, from about 5 to about 150, from about 10 to about 100, or from about 20 to about 50 inserts.

Any composition described herein according to the present disclosure can be used in a method of screening a compound for biological activity. Thus, the present disclosure also relates to a method of screening a compound for biological activity comprising exposing one or a plurality of compounds to one or a plurality of genome-edited keratinocytes according to the present disclosure. The compounds can be nucleic acid molecules, peptides, or small molecules, or any combination thereof.

EXAMPLES Example 1: Construction of Vectors

The plasmid pSpCas9(BB)-2A-GFP (Ran et al., “Genome engineering using the CRISPR-Cas9 system,” Nat. Protoc., 2013 November; 8(11):2281-308) was used to construct vectors for transfection. The plasmid comprises the following components: pUC (origin of replication; SEQ ID NO: 1), U6 promoter (guide RNA promoter; SEQ ID NO: 2), CMV promoter (Cas9-GFP promoter; SEQ ID NO: 3), Cas9 gene (SEQ ID NO: 4); GFP gene (SEQ ID NO: 5), Ampicillin promoter (SEQ ID NO; 6), Ampicillin (Bacteria selection marker; SEQ ID NO: 7), and guide RNA scaffold (SEQ ID NO: 8). The full pSpCas9(BB)-2A-GFP vector nucleotide sequence is provided in SEQ ID NO: 9. A schematic of the plasmid is shown in FIG. 4A (vector map) and 4B (linearized). This plasmid encodes for both the cas9 protein and the guide RNA(gRNA). The gRNA has a constant part (depicted in FIG. 4A and FIG. 4B as “gRNA scaffold” in the schematic) and a variable part. The variable part is the 20-nucleotide sequence that starts right at the end of the gRNA scaffold. This 20-nucleotide sequence is the one that will guide the cas9 protein to the specific site in the genome where it will create a double strand break. The 20-nucleotide sequences for each of the genes that were targeted are provided in the column entitled “Small guide RNA (5′>3′)” of Table 5.

The plasmid pSpCas9(BB)-2A-GFP was also modified to include different selection markers, including different fluorescent markers (e.g., EBFP2, Cerulean, Citrine, mKO, dTomato, and mkate2), different antibiotic resistance genes (e.g., puramycin), and different surface markers (e.g., SBP-ALNGFR).

TABLE 5 Small guide RNas and primers used for genome-editing and genome-editing assessment. Gene Small guide RNA (5′ > 3′) TIDE primers (5′ > 3′) TP53 1: CCATTGTTCAATATCGTCCG F: ACTGACCGTGCAAGTCACAG SEQ ID NO: 35 SEQ ID NO: 31 R: TGGGTTGTGGTGAAACATTG SEQ ID NO: 36 2: GAGCGCTGCTCAGATAGCGA F: CTCGATCTCCCAACCTCGTGA SEQ ID NO: 37 SEQ ID NO: 32 R: GAGGTGTAGACGCCAACTCTCTCT SEQ ID NO: 38 3: GATCCACTCACAGTTTCCAT F: TTCTGGGAAGGGACAGAAGA SEQ ID NO: 39 SEQ ID NO: 33 R: GGAAGGTTGGAAGTCCCTCT SEQ ID NO: 40 4: GGTGCCCTATGAGCCGCCTG F: CTCGATCTCCCAACCTCGTGA SEQ ID NO: 41 SEQ ID NO: 34 R: GAGGTGTAGACGCCAACTCTCTCT SEQ ID NO: 42 NOTCH1 1: TCCTGCCAGAACACCCACGG F: CATCCTCGGCTCAGTGAAGA SEQ ID NO: 47 SEQ ID NO: 43 R: AATCCGAGGACTATGAGAGCTT SEQ ID NO: 48 2: TCGCACGCCTCCTCGATCAG F: GAGCAGAGCCTTAGAACTGCAT SEQ ID NO: 49 SEQ ID NO: 44 R: GTGTGTAAAGGGTTTTGCTGCT SEQ ID NO: 50 3: TGCAGGTCAGTACTGTACCG F: GGGCATCCCGTGACACTTG SEQ ID NO: 51 SEQ ID NO: 45 R: CAGTGGGGTGTCCTGGCATT SEQ ID NO: 52 4: TTGACGTCGATCTCGCATCG F: GGACTTGGGACAGAAACGAA SEQ ID NO: 53 SEQ ID NO: 46 R: CTGTAACGAGGGCTCCAACT SEQ ID NO: 54 CDKN2A 1: CACCGAATAGTTACGGTCGG F: GACTCCCTTTTTATCCCAAACG SEQ ID NO: 59 SEQ ID NO: 55 R: CCAGTCCTCCTTCCTTGCCAAC SEQ ID NO: 60 2: CGGGTCGGGTGAGAGTGGCG F: CGGGCTGAACTTTCTGTGCT SEQ ID NO: 61 SEQ ID NO: 56 R: GGCCTAACATAATTTCAGGAAGA SEQ ID NO: 62 3: GATGATGGGCAGCGCCCGAG F: CGGGCTGAACTTTCTGTGCT SEQ ID NO: 63 SEQ ID NO: 57 R: GGCCTAACATAATTTCAGGAAGA SEQ ID NO: 64 4: GTGGCCAGCCAGTCAGCCGA F: GACTCCCTTTTTATCCCAAACG SEQ ID NO: 65 SEQ ID NO: 58 R: CCAGTCCTCCTTCCTTGCCAAC SEQ ID NO: 66 TP63 1: CAATGATTAAAATTGGACGG F: ATCCCCAGTTTTCCCTTTTC SEQ ID NO: 71 SEQ ID NO: 67 R: AGCCAGTGGATGTCTCAAGG SEQ ID NO: 72 2: GCAGTCGAGCACCGCCAAGT F: CCGTTACAAACACACACATGC SEQ ID NO: 73 SEQ ID NO: 68 R: CGTTCACGTTCTGGAGACTG SEQ ID NO: 74 3: GCTGAGCCGTGAATTCAACG F: GCAGCATGCAGCTCTAAAAA SEQ ID NO: 75 SEQ ID NO: 69 R: ACACCATTCTCCTGCCTCAG SEQ ID NO: 76 4: TGTGTGTTCTGACGAAACGC F: TCCCTACCATCCTTCCACCT SEQ ID NO: 77 SEQ ID NO: 70 R: TCCCCATTATGGACTGAAGG SEQ ID NO: 78 FAT1 1: AATTCCCAGAGATCTAGGCG F: CCAGTCGGAAGAGTGCTTCT SEQ ID NO: 83 SEQ ID NO: 79 R: CAAATAGCAATCCCCCTGAA SEQ ID NO: 84 2: ACTTGATCGAGAAACCCCGG F: ATTTCCGGTGGAAGGATTTT SEQ ID NO: 85 SEQ ID NO: 80 R: TCCATTCTGTCTGGCAATGA SEQ ID NO: 86 3: CTAACCGTTAGAGCTTCCGA F: TGCAGAAGGCTTATGTTCCTC SEQ ID NO: 87 SEQ ID NO: 81 R: CGAGCAGCTGATGCTGATAA SEQ ID NO: 88 4: CTACGACAGTCACTTCGATG F: AACGTTGGTTTGGTTTGGTT SEQ ID NO: 89 SEQ ID NO: 82 R: AAGCTTTTAAATGGCCCAGTG SEQ ID NO: 90 CASP8 1: AGGGGACTCGGAGACTGCGA F: TGCACGTCCATGAATTGTCT SEQ ID NO: 95 SEQ ID NO: 91 R: CAGGGCCAAATGAAAAAGAA SEQ ID NO: 96 2: CTACCTAAACACTAGAAAGG F: CCCATTGGCTTGTTTGTATGTC SEQ ID NO: 97 SEQ ID NO: 92 R: AAGCCTCCCAAGTGATACTGAA SEQ ID NO: 98 3: GCCTGGACTACATTCCGCAA F: CCCATTGGCTTGTTTGTATGTC SEQ ID NO: 99 SEQ ID NO: 93 R: AAGCCTCCCAAGTGATACTGAA SEQ ID NO: 100 4: TCTACTGTGCAGTCATCGTG F: CAATCGCAGGCACTCGGCA SEQ ID NO: 101 SEQ ID NO: 94 R: GGATGTACCAGGTTCCCTCTGCAG SEQ ID NO: 102 RB1 1: AAACAATCAAAGGACCGAGA F: TGGCTTTGTTGACCAAACTG SEQ ID NO: 107 SEQ ID NO: 103 R: TGCCATTCTGACTCTTGGAA SEQ ID NO: 108 2: AACATCTAATGGACTTCCAG F: CACAGATTTTTTACTGCATGGG SEQ ID NO: 109 SEQ ID NO: 104 R: GATGGCATTAATCATGGTTCC SEQ ID NO: 110 3: GGTTCTTTGACAACATGGG F: TCATACTTCAAAATCTTTCTCTGGA SEQ ID NO: 111 SEQ ID NO: 105 R: ATGCCTTTCCCCAATCCTAA SEQ ID NO: 112 4: TGAACTACTTACGAACTGCT F: GCCTCAAAAGACCACTCAGG SEQ ID NO: 113 SEQ ID NO: 106 R: TGGAGCTCTACATGGGCATA SEQ ID NO: 114 PTEN 1: AGAGCGTGCAGATAATGACA F: TCATGTGAATGAAAATGCAACA SEQ ID NO: 119 SEQ ID NO: 115 R: GAAGGCAGACAGGAGTGGAA SEQ ID NO: 120 2: AGCTGGCAGACCACAAACTG F: TTTCGTGACTACTCCCTGTTGA SEQ ID NO: 121 SEQ ID NO: 116 R: ATGCCATAAGGCCTTTTCCT SEQ ID NO: 122 3: ATTCTTCATACCAGGACCAG F: TCATGTGAATGAAAATGCAACA SEQ ID NO: 123 SEQ ID NO: 117 R: GAAGGCAGACAGGAGTGGAA SEQ ID NO: 124 4: CCAATTCAGGACCCACACGA F: TTTCGTGACTACTCCCTGTTGA SEQ ID NO: 125 SEQ ID NO: 118 R: ATGCCATAAGGCCTTTTCCT SEQ ID NO: 126 NSD1 1: AAGCACATAAAGATGAACGG F: GGAAGCCAGTGTTGGACTTG SEQ ID NO: 131 SEQ ID NO: 127 R: TTGTGGCAGCAAACCTAGAA SEQ ID NO: 132 2: GAATTGCTAGTTAAAACGCC F: GGTTCATCCACTTTTTGTAGCC SEQ ID NO: 133 SEQ ID NO: 128 R: CCCAAGTAACACAAGGATTGG SEQ ID NO: 134 3: GCCCTATCGGCAGTACTACG F: TTTTTGTTGCTGTGGGCTTA SEQ ID NO: 135 SEQ ID NO: 129 R: ACATGGCGAAATCCTGTCTC SEQ ID NO: 136 4: TATGCATGATAGTAAGACGA F: TGCAGTTTGAAATGCTGCTC SEQ ID NO: 137 SEQ ID NO: 130 R: AATGAACCTCCACGTTCTCG SEQ ID NO: 138 KMT2D 1: CCCCTATCCTAGTCCTACGG F: GGCCCATAGCTAGGAGAGGA SEQ ID NO: 143 SEQ ID NO: 139 R: CAACAAGGTGCAGAAGGTGA SEQ ID NO: 144 2: CCTTCGAAGCGAAAGTACTG F: GGGCCCTCCAGTTTAATTTC SEQ ID NO: 145 SEQ ID NO: 140 R: GTGCTGGCATTCAGCAAGTA SEQ ID NO: 146 3: GGTGGAAATTCCCGCCAACG F: GATCCTCACAGGGCAACTTC SEQ ID NO: 147 SEQ ID NO: 141 R: CCAAGGGGGATGGATTTTAT SEQ ID NO: 148 4: TTCGGGGTAGACCTCCATAG F: TAGCCCCTCACCTGTTTGAT SEQ ID NO: 149 SEQ ID NO: 142 R: CTTCCCAGTGCTCTCCTCCT SEQ ID NO: 150 AJUBA 1: AAATCGGGGGGCAACGACTG F: GTCGTAGCCCATGCTGATG SEQ ID NO: 155 SEQ ID NO: 151 R: GGCTGCAGGGAAAGAGAAC SEQ ID NO: 156 2: ACCCGACGGCTCAATCCCCG F: CATCCCTCTTGCTTCTTTCCTA SEQ ID NO: 157 SEQ ID NO: 152 R: GCTCTAGCTTCGCCAGTAGCTC SEQ ID NO: 158 3: TAGGTCCCCCCAACCCACTT F: GTCGTAGCCCATGCTGATG SEQ ID NO: 159 SEQ ID NO: 153 R: GAACAGATAGACCTGCGGACTG SEQ ID NO: 160 4: TGGACCGATTGTACGCTCAG F: CATCCCTCTTGCTTCTTTCCTA SEQ ID NO: 161 SEQ ID NO: 154 R: GCTCTAGCTTCGCCAGTAGCTC SEQ ID NO: 162 EP300 1: ATGGTGAACCATAAGGATTG F: TACCGATGGGTTTTCACCAT SEQ ID NO: 167 SEQ ID NO: 163 R: TCCTGTTTGGATCAACATGC SEQ ID NO: 168 2: CTGTAATAAGTGGCATCACG F: TTGCTGTGTTGCCTAAGCTG SEQ ID NO: 169 SEQ ID NO: 164 R: CCAAAAAGCTGTCTAGCGAGT SEQ ID NO: 170 3: GGTACGACTAGGTACAGGCG F: TTCTGTGACATAGCAATGGTGTT SEQ ID NO: 171 SEQ ID NO: 165 R: TGCTGTGCTCTGCTGTGAG SEQ ID NO: 172 4: GTGGCACGAAGATATTACTC F: TCTGGGATTGCACCACTACA SEQ ID NO: 173 SEQ ID NO: 166 R: TGAGCTGAGGCCTAGTTTTTC SEQ ID NO: 174 EPHA2 1: CACACACCCGTATGGCAAAG F: AAGACAGGGGGCATCTTTAAC SEQ ID NO: 179 SEQ ID NO: 175 R: GCGGTGTGGTGTCACTTAATAC SEQ ID NO: 180 2: CTGGTGCGGGTCAGTCCGTG F: TTGCTCTAGACACTGTGCCTTT SEQ ID NO: 181 SEQ ID NO: 176 R: CTCCTGCGAGTGTGAGGAA SEQ ID NO: 182 3: GAAGCCCCTGAAGACATACG F: ATGATGTTGTGGTGGCTGAA SEQ ID NO: 183 SEQ ID NO: 177 R: GACCAAAGTAGGGCCAGGAG SEQ ID NO: 184 4: TCACGGAGAAACCCTCGGTG F: CGGGAGGTATTGCAGGTATG SEQ ID NO: 185 SEQ ID NO: 178 R: CTGCAGATGGGGAAACTGAG SEQ ID NO: 186 FBXW7 1: AAGAGCGGACCTCAGAACCA F: AACAAGACACAGGGGCTCAT SEQ ID NO: 191 SEQ ID NO: 187 R: CACGAGGAAGGCACGTTAAA SEQ ID NO: 192 2: ACAGAATTGATACTAACTGG F: CAGAGGAGGCAGCAAATTCT SEQ ID NO: 193 SEQ ID NO: 188 R: TGTGTTTGTTTTGGGGAACC SEQ ID NO: 194 3: ACATTAGTGGGACATACAGG F: TCAAACAGGAAGCTGACAACA SEQ ID NO: 195 SEQ ID NO: 189 R: TGGTATGTAGCCACACCCAAT SEQ ID NO: 196 4: GTTGGAGTAGAACCTAGACC F: GGCTCAATTACTTTCCCTCCA SEQ ID NO: 197 SEQ ID NO: 190 R: ATTTTCCCCTGCAGAATGTG SEQ ID NO: 198 RASA1 1: AATTTAAGAACAGATGAACA F: TTACACCCCAAAACCTTCAA SEQ ID NO: 203 SEQ ID NO: 199 R: GCACGAGAAATGCTTGAACC SEQ ID NO: 204 2: ATCGCCAGGAGTATTATCTG F: AAGCAAACTGGTCTTCACTTTTT SEQ ID NO: 205 SEQ ID NO: 200 R: TCACAAAAATTGTTTTGGAAGG SEQ ID NO: 206 3: ATTTACGGAAAAGTAGTCCA F: AATCAGCACTCTCCCCCTCT SEQ ID NO: 207 SEQ ID NO: 201 R: GAACCGTTTCTGCTGTGTTG SEQ ID NO: 208 4: GGATGGACCAGAATACGAGG F: GTAACAGCCGGAGCTGGAG SEQ ID NO: 209 SEQ ID NO: 202 R: CCTCAACTTGGAAGGGACAG SEQ ID NO: 210 TGFBR2 1: ACAGTGATCACACTCCATGT F: CATGAACCCACTTCCTGACA SEQ ID NO: 215 SEQ ID NO: 211 R: TCACACAGGCAGCAGGTTAG SEQ ID NO: 216 2: ACCTACAGGAGTACCTGACG F: CATGAACCCACTTCCTGACA SEQ ID NO: 217 SEQ ID NO: 212 R: TCACACAGGCAGCAGGTTAG SEQ ID NO: 218 3: GCAGAAGCTGAGTTCAACCT F: CATGAACCCACTTCCTGACA SEQ ID NO: 219 SEQ ID NO: 213 R: TCACACAGGCAGCAGGTTAG SEQ ID NO: 220 4: TATCATGTCGTTATTAACTG F: ATCCTCTGTGCTGCCCTCTA SEQ ID NO: 221 SEQ ID NO: 214 R: TGCAGAGAACACCCCTAGAAA SEQ ID NO: 222 ZNF750 1: CAAGGGGCCAGACAACGCCG F: GGCTTCTTCAAGCAGGTGAG SEQ ID NO: 227 SEQ ID NO: 223 R: TTCCAAGTCTGTCGCAAATG SEQ ID NO: 228 2: GCGGGTACTCTGGGATTGTG F: GGCTTCTTCAAGCAGGTGAG SEQ ID NO: 229 SEQ ID NO: 224 R: TTCCAAGTCTGTCGCAAATG SEQ ID NO: 230 3: GTTCGAAAGTCCAATTCCTG F: TGGAACCCAACCAGAAACAT SEQ ID NO: 231 SEQ ID NO: 225 R: CCATTTCCCCTTACATGCAC SEQ ID NO: 232 4: TCTGGCCTGTAAAATCCGTA F: TGGAACCCAACCAGAAACAT SEQ ID NO: 233 SEQ ID NO: 226 R: CCATTTCCCCTTACATGCAC SEQ ID NO: 234 KEAP1 1: ACAACCCCATGACCAATCAG F: AGGAACCATATGGGTTTGTGAC SEQ ID NO: 239 SEQ ID NO: 235 R: TCAACTGGGTCAAGTACGACTG SEQ ID NO: 240 2: AGTACGACTGCGAACAGCGA F: GAGCGACTGTCGGAAGTAGCC SEQ ID NO: 241 SEQ ID NO: 236 R: CTTCAGCCTCCCGAGTAGCTG SEQ ID NO: 242 3: CAGCACCGTTCATGACGTGG F: CCCCTCAAACTGTGGAGACT SEQ ID NO: 243 SEQ ID NO: 237 R: TATCTTCTGGAACCCCATGC SEQ ID NO: 244 4: CCTGGAGGATCATACCAAGC F: CCAATCTGCTCAGCGAAGTT SEQ ID NO: 245 SEQ ID NO: 238 R: GCCAATCAGGTTGATCAGGT SEQ ID NO: 246 HRAS 1: CCAGGCTCACCTCTATAGTG F: ATGGCAAACACACACAGGAA SEQ ID NO: 251 SEQ ID NO: 247 R: TTTTCCCATCACTGGGTCAT SEQ ID NO: 252 2: GGACTCGGATGACGTGCCCA F: GACACTCTGGGGACAAGAGG SEQ ID NO: 253 SEQ ID NO: 248 R: GTTGGACATCCTGGATACCG SEQ ID NO: 254 3: TGACCTGGCTGCACGCACTG F: GACACTCTGGGGACAAGAGG SEQ ID NO: 255 SEQ ID NO: 249 R: GTTGGACATCCTGGATACCG SEQ ID NO: 256 4: TGTTGATGGCAAACACACAC F: CAAGGGAGAGGGTCAGTGAG SEQ ID NO: 257 SEQ ID NO: 250 R: CCAGGACACAGCCAGGATAG SEQ ID NO: 258 PIK3CA 1: AACCTCGAACCATAGGATCT F: CCCCGAAATTCTACCCAAAT SEQ ID NO: 263 SEQ ID NO: 259 R: AAAAGTGCCCAATCCTTTGA SEQ ID NO: 264 2: GAAGCTGTATAATGCTTGGG F: GGCCGTGATCCTTGTTTTTA SEQ ID NO: 265 SEQ ID NO: 260 R: AGGTCTTTCTGCCAAACGAA SEQ ID NO: 266 3: GGATTTAGCTATTCCCACGC F: TGGGGAAGAAAAGTGTTTTGA SEQ ID NO: 267 SEQ ID NO: 261 R: TCCATGTTGTTCAAGGGTCA SEQ ID NO: 268 4: GTTCGAACAGGTATCTACCA F: ACTGCTTCACCAGCTAAGGA SEQ ID NO: 269 SEQ ID NO: 262 R: CTCAGCCTTCCAGGTTCAAG SEQ ID NO: 270 RHOA 1: AAAACACATCAGTATAACAT F: TGTTGCTCAGTCTGGAATGC SEQ ID NO: 275 SEQ ID NO: 271 R: GGGCGACAGAGCAAGACTAC SEQ ID NO: 276 2: ACAGAAATGCTTGACTTCTG F: TTGCAGTGAGCTGAGATTGC SEQ ID NO: 277 SEQ ID NO: 272 R: CCCCGTGAAACTACCTTGTG SEQ ID NO: 278 3: CTATGTGGCAGATATCGAGG F: ACATGCCTGTAATCCCTGCT SEQ ID NO: 279 SEQ ID NO: 273 R: GAACTGAGATTGGGCCAGTG SEQ ID NO: 280 4: GCCACTCACCTAAACTATCA F: TGTTGCTCAGTCTGGAATGC SEQ ID NO: 281 SEQ ID NO: 274 R: GGGCGACAGAGCAAGACTAC SEQ ID NO: 282 CCR5 1: CAATGTGTCAACTCTTGACA F: CTCCGCTCTACTCACTGGTGTTC SEQ ID NO: 287 SEQ ID NO: 283 R: GACAAAGGCATAGATGATGGGGTTG SEQ ID NO: 288 2: CATTAAAGATAGTCATCTTG F: CTCCGCTCTACTCACTGGTGTTC SEQ ID NO: 289 SEQ ID NO: 284 R: GACAAAGGCATAGATGATGGGGTTG SEQ ID NO: 290 3: GGTGACAAGTGTGATCACTT F: CTCCGCTCTACTCACTGGTGTTC SEQ ID NO: 291 SEQ ID NO: 285 R: GACAAAGGCATAGATGATGGGGTTG SEQ ID NO: 292 4: TCATCCTCCTGACAATCGAT F: CTCCGCTCTACTCACTGGTGTTC SEQ ID NO: 293 SEQ ID NO: 286 R: GACAAAGGCATAGATGATGGGGTTG SEQ ID NO: 294 ROSA26 1: GTTGCTTTATCACTCAACAG F: TGCCATCATAGTGAACTACAGCCT SEQ ID NO: 298 SEQ ID NO: 295 R: GGGAGGATGTGTGTAAGTTGTATGC SEQ ID NO: 299 2: GGAATGATCTTGAATAGTGT F: TGCCATCATAGTGAACTACAGCCT SEQ ID NO: 300 SEQ ID NO: 296 R: GGGAGGATGTGTGTAAGTTGTATGC SEQ ID NO: 301 3: AATGCAGGTATGCCACTACT F: TGCCATCATAGTGAACTACAGCCT SEQ ID NO: 302 SEQ ID NO: 297 R: GGGAGGATGTGTGTAAGTTGTATGC SEQ ID NO: 303 AAVS1 1: AATGAATTGATGCAGGTCTG F: GAGCCAAAGTTAGAACTCAGGACC SEQ ID NO: 305 SEQ ID NO: 304 R: ACCTCTCACTCCTTTCATTTGG SEQ ID NO: 306

Example 2: Isolation and Culture of Primary Cells

Primary adult human epidermal and oral keratinocytes were isolated from fresh surgical specimens. Primary newborn foreskin keratinocytes were acquire from the department of dermatology core service (UCSF). Surgical samples were incubated in dispase (Corning, #354235) overnight, after which epidermis was physically separated from dermis using sterilized tweezers. To dissociate the epidermis, 0.5% trypsin (Life Techonologies, #15400054) and 3 minutes incubation was used. Cells were plated in 6-well plate using keratinocyte media. Keratinocyte media was composed by keratinocyte-SFM (Life technologies, #17005042) and 154 medium (Life techcnologies, #M154500) in a 1:1 ratio, supplemented with human keratinocyte growth supplement (HKGS, Life technologies, #S0015). Primary keratinocytes were grown at 37° C., 5% CO2. Cells were monitored periodically and split before cultures reaching 90% confluency using 0.05% trypsin (Life technologies, #25300054).

Primary adult fibroblast were isolated from the dermis produced in the keratinocyte isolation protocol. Collagenase P (Sigma-Aldrich, #11213857001) and dispase (Corning, #354235) were used to dissociate fibroblasts. Fibroblasts were grown in DMEM (Life Technologies, #12491023) supplemented with 10% fetal bovine serum (FBS, Fisher Scientific, #MT35010CV) and 1× penicillin/streptomycin (Fisher Scientific, #MT30002CI) at 37 C, 5% CO₂.

Example 3: Primary Cells Transfection

Keratinocytes were seeded on tissue culture-treated polystyrene 6-well plates (Corning, #0877249) at a density of 150,000 cells per well. 3 μg of vector DNA and 4.5 μL of TranslT-X2 (Mirus Bio, #mir6000) was used per transfection. In the case of multiple gene targeting experiments, the total amount of vector DNA (3 μg) was created by combining different vectors encoding for distinct guide RNAs in a 1:1 ratio. Cells were incubated with transfection complexes for 24 hours at 37° C., 5% CO2.

Alternatively, keratinocytes were transfected by electroporatioin using, for example, a Lonza 4D-Nucleofector X Unit (Lonza, #AAF-1002X). To this end, CRISPR/Cas9 ribonucleoprotein (RNP) complexes were formed by mixing crRNA:tracrRNA (50 μM) and Cas9 protein (40 μM) at a ratio 16:10 and subsequently incubated for 15 minutes at room temperature. An example of tracrRNAs may have the sequence (5^(mut) to 3′) of GTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG AGTCGGTGC (SEQ ID NO: 346). The guide RNAs are comprised in the crRNA molecules used to form CRISPR/Cas9 RNP complexes. TracrRNA molecules were tagged with the fluorescent molecule ATTO 550 (Integrated DNA technologies, #1075928) for transfection efficiency assessment. CRISPR/Cas9 RNPs complexes were added to 500,000 keratinocytes, resuspended in nucleofector solution (Lonza, # V4XP-3024), at a final concentration of 1 μM (final volume 25 μL). RNPs targeting the individual genes were prepared using specific crRNA molecules and, if combined, final concentration for each target was kept at 1 μM. For transfections with plasmid DNA, a total amount of 3 μg per target was added to the cells. Electroporation was carried out using the Lonza 4D-Nucleofector X Unit (Lonza, #AAF-1002X) and suggested protocol for Amaxa™ 4D-Nucleofector™ Protocol for Normal Human Epidermal Keratinocytes (NHEK) kit (Lonza, # V4XP-3024). Cells were pipetted into 6-well plates and kept in the incubator (37° C., 5% CO₂) afterwards.

Example 4: Primary Cells Selection and Expansion

Keratinocytes transfected with pSpCas9(BB)-2A-GFP vectors show GFP fluorescence levels within few hours. FIG. 5 shows the percentage of GFP positive cells detected after up to 64 hours post-transfection. The percentage of GFP positive cells observed at 24 hours post-transfection appeared to be comparable to that observed at 40 hours post-transfection, which is the highest amongst all the time points tested. The 24-hour post-transfection was therefore chosen for cell harvest. FIG. 6 shows the percentage of primary keratinocytes showing genome-editing 24 hours after transfection, which evidences the capacity of the method according to the present disclosure to knock out these genes in primary keratinocytes.

Twenty-four hours after transfection, cells were harvested and dissolved in sorting buffer (5% FBS, 5 mM EDTA (Invitrogen, # AM9261) in PBS (Life Technologies, # AM9624)). Transfected cells were selected by Fluorescence-Activated Cell Sorting (FACS). GFP fluorescence intensity was determined by the SONY SH800 sorter and software. Transfected keratinocytes were sorted onto tissue culture-treated polystyrene 96-well plates (Corning, #CLS3595) and cultures were expanded. Genome-editing in transfected keratinocytes was checked by a method named TIDE for Tracking of Indels by Decomposition (Brinkman et al., Easy quantitative assessment of genome editing by sequence trace decomposition, Nucleic Acids Res., 2014 Dec., 16; 42(22):e168; accessible at deskgen.com/landing/tide.html#/tide). The TIDE method, which requires only a pair of PCR reactions and two standard capillary sequencing runs, can deconvolute Sanger capillary sequencing reactions to infer the proportion of a population of cells whose genome has been edited. The primers for genome-editing assessment using this TIDE method are provided in the column entitled “TIDE primers (5′>3′)” of Table 5. FIG. 7 shows the genome editing efficiency measured by TIDE before and after FACS selection. As shown in FIG. 7 , the genome editing efficiency increased from 1.8% to 99.1% by way of the FACS selection.

The behavior of genome-edited keratinocytes against untreated (untransfected) cells was also analyzed, the results of which may give a hint of the effect of the individual gene knock-out (KO). FIG. 8 shows the percentage of genome-edited keratinocytes in multiple conditions (in each condition, one single gene was targeted with CRISPR/Cas9) over different passages. In the majority of the conditions, the genome-edited population disappears, while in others, such as KEAP1 KO cells or EP300 KO cells, the KO cells seem to take over the untreated population. In some conditions, such as RASA1, the percentages of edited and unedited population remain constant.

The viability of sorted genome-edited primary keratinocytes (pure populations) in vitro was also analyzed. FIG. 9 shows the recovery of genome-edited keratinocytes harboring one single mutation in the genome following FACS-selection. Among the genes analyzed, only CDKN2A KO cells could be recovered and expanded, while CASP8 KO and TGFBR2 KO cells recovered temporally, but after few doublings these cells become senescent.

Example 5: Stand Preparation for Three-Dimensional Organotypic Culture

Stands were design using the online tool TINKERCAD. The final design is a 10 by 10 mm hollow cube with walls on the sides, and an internal rim that leaves an opening of 5 by 5 mm on the bottom. 2 mm legs were included on all the bottom corners to increase the volume of media to be used in the organotypic cultures. A schematic representation of a stand design, including the 3D view, top view, side view, and front view, is provided in FIG. 10 . FIG. 11 further shows the ratio of ledge surface and cavity area of a stand from a top view. This stand design provides 56 mm² of ledge surface and 25 mm² of cavity area, thus a ledge surface/cavity area ratio of 2.24. Stands were printed with autoclavable ABS plastic using an “Objet Alaris Desktop 3D Printer.” Stands were sterilized using an standard autoclave.

Example 6: Three-Dimensional Organotypic Culture

Dermis section was obtained from discarded surgical samples and cadaver skin, and sections were frozen at −80° C. Dermis was thawed and pre-sterilized/decellularized in PBS (Life-Technologies, #AM9624) containing 1% penicillin/streptomycin (Fisher-Scientific, #MT30002CI) and 1% antibiotic-antimycotic (Life-Technologies, #15240062). Dermis was sterilized by exposing it to gamma-radiation for 100 minutes in a Cesium Radiator. Dermis can also be sterilized by incubating in 0.1% peracetic acid (PAA) for 30 minutes followed by washing 3 times with PBS for 1 hour at room temperature.

Prior to fibroblast re-impregnation, dermis was sectioned (500 microns or thinner) with a scalpel in 8 by 8 mm pieces, which were air-dried at room temperature for 3 hours in tissue culture-treated 24 well plates (epidermal side facing down). 25,000 human primary fibroblasts in 1 mL of fibroblast media were reintroduced into each of the dermis pieces by centrifugation (1 hour, 200g). Re-impregnated dermis was incubated for 7 days at 37° C., 5% CO2.

Dermis was flipped and mounted onto 3D-printed stands using sterilized tweezers. A thin layer of matrigel (25-50 μL; Corning, #354235) was applied to the bottom of each of the stands, which were placed in a tissue-culture treated 24 well-plate. 200,000 keratinocytes were resuspended in 50 μL of keratinocyte media and seeded on top of the basal membrane of the dermis. Approximately 500 μL of organotypic culture media was added to the wells in order to create an air-liquid interface. FIG. 12 illustrates this whole process. Organotypic culture media is composed of:

-   -   DMEM/HAM F12(3:1; Life Technologies, #12491023; VWR, #12-615F)         supplemented with 10% FBS (Fisher Scientific, #MT35010CV)     -   1% penicillin/streptomycin (Fisher-Scientific, #MT30002CI)     -   1% antibiotic-antimycotic (Life-Technologies, #15240062)     -   3 μg/mL of adenine (Sigma, #A9795)     -   3 ng/mL of cholera toxin (EMD Millipore, #227036)     -   40 ng/mL of hydrocortisone (EMD Millipore, #386698)     -   5 μg/mL of insulin (Sigma, #I1882)     -   10 ng/mL of EGF (Life Technologies, #PHG0315)     -   5 μg/mL of Transferrin (Sigma, #T0665)     -   1.5 ng/mL of triiodo-L-thyronine (Sigma, #T2877)     -   10 ng/mL of ciprofloxacin hydrochloride (Sigma, #PHR1044).         Organotypic cultures were incubated for 7 days at 37 C, 5% CO2.

Fixation was achieved by immersing the whole culture (including stand) in 4% PFA (Electors microscopy sciences, #15710) overnight. Organotypic cultures was embedded in OCT (Fisher-Scientific, #4585) following incubations in 15%, 30% and 50% sucrose solutions (Fisher Scientific, #50712768). In order to analyze the structure of the organotypic cultures, blocks were sectioned vertically and 5 μm frozen sections were stained using standard H&E or immunofluorescence protocols.

Example 7: Genome-Edited Keratinocytes on Organotypic Culture

Genome-edited keratinocytes containing single or multiple mutations of the genes listed in Table 1 were generated and tested on organotypic cultures as described above. The structure of the organotypic cultures of these genome-edited keratinocytes were sectioned and stained using standard H&E or immunofluorescence protocols, and the results are shown in FIG. 13 . Invasion in keratinocytes containing a combination of TP53, CDKN2A and NOTCH1 mutations were readily detectable within a week. When compared to the stain image obtained from squamous cell carcinoma tumor (FIG. 14A), these TP53+CDKN2A+NOTCH1 mutated keratinocytes appeared to have a similar structure (FIG. 14B). These results indicate that the cells were turned into cancer cells, making it as an authentic genetic model in squamous cell carcinoma.

Example 8: Minimal Mutational Determinants of Human Squamous Cell Carcinoma

Chronic exposure of squamous epithelia to carcinogenic agents such as UV radiation (1), tobacco smoke, and alcohol (2), or infection by the human papillomavirus (HPV) (3), promotes the development of dysplastic lesions that ultimately progress to squamous cell carcinomas (SCCs). SCCs are among the most prevalent human cancer types and can arise from multiple distinct tissues in the human body (4). It is believed that the sequential accumulation of mutations in relevant epithelial cells is responsible for enabling SCC formation. Although there are promising new therapeutic modalities (5), surgery and radiotherapy with or without classical chemotherapy remain the mainstay of treatment for most types of SCC, and few significant improvements in treatment outcomes have occurred at the population level in the past 40 years (6).

SCC displays a striking commonality of recurrent genomic alterations, despite distinct anatomic sites of origin (7-13). Thus, the molecular signals that govern carcinogenesis are likely shared. In HPV negative head and neck SCC (HNSCC), for example, mutations in the tumor suppressor gene TP53 are found in 85% of the cases. Inactivation of the cyclin-dependent kinase inhibitor CDKN2A occurs in the majority of these cases via point mutation, deletion or methylation-mediated gene silencing (14). Other putative driver mutations have been identified at lower frequencies (14), but validation of their pathogenic role has not been performed in most cases. Despite a high mutation burden (15) and frequent genome instability found in SCC tumors (16), no individual events have been reported to be sufficient or strictly required to trigger SCC. Sequencing studies performed on phenotypically normal skin and esophageal epithelium have shown that apparent driver mutations accumulate in aging keratinocytes without resulting in malignant lesions (17, 18). These mutations resemble those found in SCC, suggesting that specific combinations but not individual genomic alterations are required to transform normal epithelial tissue; however, the specific combinations that are sufficient to do so are currently unknown.

This study is aimed to identify the minimal combination of genetic alterations sufficient for transformation of human keratinocytes by engineering the most common putative driver mutations found in SCC into primary human keratinocytes, in both an individual and combinatorial fashion. It was found that mutations in single driver genes are not sufficient for keratinocyte malignant transformation. In contrast, the combination of TP53 and CDKN2A mutations is sufficient to immortalize keratinocytes and induce dysplastic lesions in organotypic skin and oropharyngeal mucosa cultures. Moreover, keratinocytes harboring mutations in TP53, CDKN2A, and NOTCH1 exhibit an SCC-like phenotype, including invasion through the native dermal basement membrane. Transcriptomic analyses demonstrate that NOTCH1 mutations induce an epithelial to mesenchymal transition (EMT) like phenotype in TP53^(mut) CDKN2A^(mut) human primary keratinocytes while obstructing keratinocyte differentiation.

1. Materials and Methods

i. Isolation, Culture, Genome-Editing and Selection of Primary Keratinocytes

Isolation and culture of primary cells were performed as described in Example 2. Briefly, primary adult human epidermal and oral tonsil keratinocytes were isolated from fresh surgical specimens. Surgical samples were incubated in dispase (Corning, #354235) overnight, after which epidermis was physically separated from dermis using sterilized tweezers. 0.5% trypsin (Life Technologies, #15400054) was used to dissociate the epidermis. Cells were plated in a 6-well plate using keratinocyte media. Keratinocyte media was prepared with keratinocyte-SFM (Life technologies, #17005042) and 154 medium (Life techcnologies, #M154500) in a 1:1 ratio, supplemented with human keratinocyte growth supplement (HKGS, Life Technologies, #S0015). Primary keratinocytes were grown at 37° C., 5% CO2. Cells were monitored periodically and split before cultures reached 90% confluence.

Primary adult fibroblasts were isolated from the dermis produced in the keratinocyte isolation protocol. Collagenase P (Sigma-Aldrich, #11213857001) and dispase (Corning, #354235) were used to dissociate fibroblasts. Fibroblasts were grown in DMEM (Life Technologies, #12491023) supplemented with 10% fetal bovine serum (FBS, Fisher Scientific, #MT35010CV) and 1× penicillin/streptomycin (Fisher Scientific, #MT30002CI) at 37° C., 5% CO2.

The genome of primary keratinocytes was edited via CRISPR/cas9 system (35) as described in Example 1. pSpCas9(BB)-2A-GFP plasmids were modified to include guideRNAs to target different genes (Table 5). Transfection of primary keratinocytes was formed as described in Example 3. Briefly, primary keratinocytes were transfected using 3 μg of DNA plasmids and 4.5 μL of TranslT-X2 (Mints Bio, #mir6000). Incubation for 30 minutes at room temperature prior to treatment was necessary for the formation of transfecting complexes. In the case of multiple gene targeting experiments, the total amount of DNA (3 μg) resulted from the combination of different vectors encoding for distinct guideRNAs in a 1:1 ratio. Cells were incubated with transfection complexes for 24 hours at 37° C., 5% CO2. Ubiquitous genes such as CCR5 and ROSA26 were used as control targets in the genome-editing experiments. A multitude of different guide RNA molecules were developed and used to confirm which specific mutation was responsible for a phenotype. Table 5 contains the list of guideRNAs used in these experiments.

Primary keratinocytes selection and expansion were performed as described in Example 4. Briefly, 24 hours after transfection, keratinocytes were selected by fluorescence-activated cell sorting (FACS) onto 96-well plates (Corning, #CLS3595) using a Sony SH800S cell sorter.

In cell proliferation assays, cells from each condition were seeded in 24-well plates. Cells were counted from three different wells each day, and the plotted value represents the mean. Keratinocytes were stained with DyeCycle Violet (Life technologies, #V35003) following manufacturer's recommendations followed by FACS for cell cycle analysis (10,000 cells per condition). Unpaired t tests (two-tailed) were used in cell proliferation and cell cycle analyses to assess statistical significance.

ii. Genomic DNA Extraction, PCR and Sequencing

Genomic DNA was isolated using the QIAamp Mini Kit (Qiagen, #56304) following manufacturer's instructions. PCR reactions were performed using Q5 High fidelity DNA polymerase (New England Biolabs, #M0491). PCR bands were extracted using NucleoSpin Gel and PCR clean-up columns (Takara, #740609) and sequenced using PCR primers. Genome-editing in transfected keratinocytes was assessed by TIDE (19) as described in Example 4 using the primers for genome-editing assessment provided in the column entitled “TIDE primers (5′>3′)” of Table 5. A diagram representing keratinocyte transfection and sorting based on GFP expression is shown in FIG. 3 . TIDE results showing genome-editing efficiency (percentage of cells harboring mutations in the FAT1 gene loci) for non-sorted (top) and sorted (bottom) populations is shown in FIG. 7 . Statistical analysis (P-value) associated to each condition was calculated with TIDE (based on two-tailed t-test of the variance-covariance matrix of the standard errors).

Targeted next-generation sequencing of primary keratinocytes was performed using the UCSF500 Cancer Panel as previously described (36, 37). Capture-based next-generation DNA sequencing was performed using an assay that targets a total footprint of ˜2.9 Mb, which includes all coding exons of 479 cancer-related genes, and select introns and upstream regulatory regions of 47 genes to enable detection of TERT promoter variants and structural variants including gene fusions (Table 6). The assay also includes baits for 2,000 unique DNA segments containing common single nucleotide polymorphisms (SNPs) within regions devoid of constitutional copy number variants to enable genome-wide copy number and zygosity analysis. Multiplex library preparation was performed using the KAPA Hyper Prep Kit (Roche, Santa Clara, Calif.) according to the manufacturer's specifications. Hybrid capture of pooled libraries was performed using a custom oligonucleotide library (NimbleGen SeqCap EZ Choice, Roche, Basel, Switzerland). Captured libraries were sequenced as paired-end 100 bp reads on a HiSeq 2500 instrument (Illumina, San Diego, Calif.). Sequence reads were mapped to the reference human genome build GRCh37 (hg19) using the Burrows-Wheeler aligner (bio-bwa.sourceforge.net/). Recalibration and deduplication of reads was performed using the Genome Analysis Toolkit (gatk.broadinstitute.org/hc/en-us). Coverage and sequencing statistics were determined using Picard CalculateHsMetrics and Picard CollectlnsertSizeMetrics. Single nucleotide variant and insertion/deletion mutation calling was performed with Unified Genotyper and Delly. Variant annotation was performed with Annovar. Single nucleotide variants, insertions/deletions, and structural variants were visualized and verified using Integrated Genome Viewer. Genome-wide copy number analysis based on on-target and off-target reads was performed by CNVkit and visualized using Nexus Copy Number (Biodiscovery, El Segundo, Calif.).

TABLE 6 Targeted gene panel-List of the 479 genes targeted for sequencing on the UCSF500 Cancer Panel. In addition to the coding exons, select intronic or upstream regulatory regions are also captured for genes highlighted in bold. ABL1 CD274 EP300 GATA2 KMT2D NRAS RAD21 SPRY2 ABL2 CDC42 EPCAM GATA3 KNSTRN NSD1 RAD50 SPRY4 ACVR1 CDC73 EPHA2 GLI1 KRAS NSD2 RAD51 SPTA1 ACVR1B CDH1 EPHA3 GLI2 LEF1 NT5C2 RAD51C SRC AJUBA CDK12 EPHA5 GNA11 LIFR NTRK1 RAD51D SRSF2 AKT1 CDK4 EPHA7 GNA13 LRP1B NTRK2 RAF1 SS18 AKT2 CDK6 EPHBI GNAQ LZTR1 NTRK3 RARA STAG2 AKT3 CDK8 EPOR GNAS MALAT1 NUP93 RASA1 STAT3 ALK CDKN1A ERBB2 GPC3 MAML2 NUTM1 RASA2 STAT4 APC CDKN1B ERBB3 GPR124 MAP2K1 OR5L1 RB1 STAT6 APOBEC3G CDKN2A ERBB4 GRIN2A MAP2K2 PAK1 RBM10 STK11 AR CDKN2B ERCC1 GRM3 MAP2K4 PAK3 REL SUFU ARAF CDKN2C ERCC2 GSK3B MAP3K1 PALB2 RELA SYK ARFRP1 CEBPA ERG H3F3A MAP3K2 PARK2 RET SYNE1 ARHGAP35 CHD1 ERRFI1 H3F3B MAP3K5 PAX3 RHEB TADA1 ARID1A CHD2 ESPL1 HDAC4 MAP3K7 PAX5 RHOA TBX3 ARID1B CHD4 ESR1 HDAC9 MAP3K9 PAX7 RICTOR TCEB1 ARID2 CHD5 ESR2 HEY1 MAPK1 PAX8 RIT1 TCF7L2 ARID5B CHEK1 ETS1 HGF MCL1 PBRM1 RNF43 TERT ASH2L CHEK2 ETV6 HIF1A MDM2 PDCD1LG2 ROBO1 TET2 ASXL1 CIC EWSR1 HIST1H3B MDM4 PDGFB ROS1 TFE3 ASXL2 CLDN18 EZH1 HMGA2 MED12 PDGFRA RPL10 TFEB ATF1 CNOT3 EZH2 HNF1A MEF2B PDGFRB RPTOR TGFBR2 ATM COL1A1 FAM123B HOXB13 MEN1 PDK1 RRAGC TLR4 ATR COL2A1 FAM46C HRAS MET PHF6 RRAS TNFAIP3 ATRX CRCT1 FANCA HSPA2 MGA PHOX2B RRAS2 TNFRSF14 AURKA CREB1 FANCC HSPA5 MGMT PIK3CA RSPO2 TOP1 AURKB CREBBP FANCE HSP90AB1 MITF PIK3CG RSPO3 TOP2A AXIN1 CRKL FANCF ID3 MLH1 PIK3R1 RUNX1 TMPRSS2 AXIN2 CSF1R FANCG IDH1 MLH3 PIK3R2 RUNX1T1 TP53 AXL CSF3R FANCL IDH2 MPL PLAG1 SDHB TRAF3 BAP1 CTCF FAT1 IGF1R MRE11A PLCB4 SDHD TRAF7 BARD1 CTNNA1 FAT3 IGF2 MSH2 PMS1 SETBP1 TRIM28 BCL2 CTNNB1 FBXW7 IGF2R MSH3 POLD1 SETD2 TSC1 BCL2A1 CUL3 FGF10 IKBKE MSH6 POLE SF3B1 TSC2 BCL2L1 CUX1 FGF14 IKZF1 MTOR POLQ SH2B3 TSHR BCL2L12 CYLD FGF19 IKZF2 MUTYH POT1 SHH TSHZ2 BCL2L2 CXCR4 FGF23 IKZF3 MYB POU3F2 SIN3A TSHZ3 BCL6 DCC FGF3 IL2RB MYBL1 PPM1D SLIT2 TSLP BCOR DDIT3 FGF4 IL7R MYC PPP2R1A SLITRK6 TTYH1 BCORL1 DDR2 FGF6 INHBA MYCL1 PPP6C SMAD2 TYK2 BLM DDX3X FGFR1 INPP4B MYCN PRDM1 SMAD3 U2AF1 BRAF DDX41 FGFR2 IPMK MYD88 PREX2 SMAD4 USP7 BRCA1 DGKH FGFR3 IRF4 MYH9 PRKACA SMARCA2 VEGFA BRCA2 DICER1 FGFR4 IRS2 NAV3 PRKAG2 SMARCA4 VHL BRD4 DIS3 FH JAK1 NBN PRKAR1A SMARCB1 WISP3 BRIP1 DNAJB1 FLCN JAK2 NCKAP5 PRKCA SMC1A WRN BTG1 DNMT3A FLT1 JAK3 NCOA2 PRKCH SMC3 WT1 BTK DOT1L FLT3 JAZF1 NCOA3 PRKDC SMO XBP1 CALR DUSP2 FLT4 KAT6A NCOR1 PTCH1 SNCAIP XPO1 CARD11 DUSP4 FOXA1 KDM5A NF1 PTCH2 SOCS1 YAP1 CBFB DUSP6 FOXL2 KDM5C NF2 PTEN SOS1 YWHAE CBL DYNC1I1 FOXO1 KDM6A NFE2L2 PTK2B SOS2 ZBTB20 CBLB EBF1 FOXP1 KDR NFKBIA PTPN1 SOX9 ZFHX3 CCND1 EDNRB FRS2 KEAP1 NFKBIE PTPN11 SOX10 ZMYM3 CCND2 EGFR FUBP1 KIT NIPBL PTPRB SOX2 ZNF217 CCND3 EGRI FUS KLF4 NKX2-1 PTPRD SPEN ZNF703 CCNE1 EIF1AX FYN KLHL6 NOTCH1 PTPRK SPOP ZNFHX4 CD79A ELF3 GAB2 KMT2A NOTCH3 PTPRT SPRED1 ZRSR2 CD79B EMSY GATA1 KMT2B NPM1 RAC1 SPRY1

i. Stand Preparation for Tree-Dimensional Organotypic Culture

Stands for three-dimensional organotypic culture were prepared according to Example 5. A schematic representation of the stand design, including the 3D view, top view, side view, and front view, is provided in FIG. 10 with the ratio of ledge surface and cavity area of the stand from a top view shown in FIG. 11 . Stands were printed with autoclavable ABS plastic using an “Objet Alaris Desktop 3D Printer.” Stands were sterilized using a standard autoclave prior their use for tissue culture.

ii. Tree-Dimensional Organotypic Culture

Three-dimensional organotypic culture was performed as described in Example 6. Briefly, dermis was obtained from discarded surgical samples and cadaver skin, and sections were frozen at −80° C. Dermis was thawed and pre-sterilized/decellularized in PBS (Life-Technologies, #AM9624) containing 1% Penicillin/Streptomycin (Fisher-Scientific, #MT30002CI) and 1% antibiotic-antimycotic (Life-Technologies, #15240062). Dermis was sterilized using gamma-radiation or 0.1% Peracetic Acid treatment for 3 hours (Sigma-Aldrich, #269336).

Prior to fibroblast re-impregnation, dermis was sectioned with a scalpel in 8 by 8 mm pieces, which were air-dried at room temperature for 3 hours (epidermal side facing down). Twenty-five thousand (25,000) human primary fibroblasts in 1 mL of fibroblast media were reintroduced into each of the dermis pieces by centrifugation (1 hour, 200 g). Cell counts were performed with a Bio-Rad TC20 cell counter (#1450102). Re-impregnated dermis was incubated for 7 days at 37° C., 5% CO2.

Dermis was flipped and mounted onto 3D-printed stands using sterilized tweezers. A thin layer of Matrigel (25-50 μL; Corning, #354235) was applied to the bottom of each of the stands. Two hundred thousand (200,000) keratinocytes in 50 μL of keratinocyte media were seeded on top of the basal membrane of the dermis. Approximately, 500 μL of organotypic culture media was added to the wells in order to create an air-liquid interface (FIG. 12 ). Organotypic culture media is composed of DMEM/HAM F12 (3:1; Life Technologies, #12491023; VWR, #12-615F) supplemented with 10% FBS (Fisher Scientific, #MT35010CV), 1% Penicillin/Streptomycin (Fisher-Scientific, #MT30002CI), 1% antibiotic-antimycotic (Life Technologies, #15240062), 3 μg/mL of adenine (Sigma, #A9795), 3 ng/mL of cholera toxin (EMD Millipore, #227036), 40 ng/mL of hydrocortisone (EMD Millipore, #386698), 5 μg/mL of insulin (Sigma, #I1882), 10 ng/mL of EGF (Life Technologies, #PHG0315), 5 μg/mL of Transferrin (Sigma, #T0665), 1.5 ng/mL of triiodo-L-thyronine (Sigma, #T2877) and 10 ng/mL of ciprofloxacin hydrochloride (Sigma, #PHR1044). Organotypic cultures were incubated for 7 days at 37° C., 5% CO2.

Fixation was achieved by immersing the whole culture (including stand) in 4% PFA (Electron Microscopy Sciences, #15710) overnight. Organotypic cultures was embedded in OCT (Fisher-Scientific, #4585) following incubations in 15%, 30% and 50% sucrose solutions (Fisher Scientific, #50712768). In order to analyze the structure of the organotypic cultures, blocks were sectioned vertically and 5 μm frozen sections were stained using standard H&E or immunofluorescence protocols.

iii. Immunohistochemistry (IHC) Staining

IHC was performed following standard protocol. Briefly, slides were incubated in Hematoxylin solution (ThermoFisher Scientific, #22220101) for 5 minutes. Following water washes, slides were incubated in Eosin (ThermoFisher Scientific, #22220104) solution for 1 minute. After a 3 minute wash in water, slides were dehydrated successively in 70%, 90% and 100% EtOH for 30 seconds. Slides were mounted with Cytoseal (ThermoFisher Scientific, #22050262) and allowed to dry for 24 hours. Tissue sections were submitted for pathology review.

Histology IHC stains were performed on an Automated Leica BOND III slide stainer. The stainer applies antigen retrieval (30 minutes) and antibody reagents according to validated protocols. Antibodies used in this study include: P40 (undiluted, Biocare, #BC28), KI67 (1:50 dilution, Dako, #MIB1).

iv. Extreme Limiting Dilution Assay

Extreme limiting dilution analysis (ELDA) was performed as previously described Briefly, primary keratinocytes harboring different combinations of mutations were collected from 10 cm dishes and diluted into single cell suspension and plated at concentrations of 1,000 to 1 cells per 100 μl of keratinocyte media in 96-well ultra-low attachment plates (Costar Corning, #3474). Cells were incubated at 37° C. in a 5% CO₂ for 10 days. At the end of 10 days, the number of wells showing spheres were counted by light microscope. ELDA web tool (bioinf.wehi.edu.au/software/elda) was used to determine frequencies of sphere forming cells and the statistical significance.

v. RNA Sequencing and Analysis

RNA was isolated using the RNAeasy Plus Kit (Qiagen, #74136). Sequencing libraries were prepared with QuantSeq 3^(mut) mRNA-Seq Library Prep Kit (Lexogen, #015.24). Sequencing was performed using a HiSeq 4000 sequencer (SE, 50 bp) and results were processed following standard ENCODE guidelines using Bluebee's online platform. Differential gene expression analysis and statistical analysis was performed using the DESeq2 package (39). GO terms and pathways analyses were performed with Panther Classification System (40, 41). Primary RNAseq data will be available upon publication in GEO.

vi. In vivo experiments

Injections of genome-engineered keratinocytes were performed subcutaneously in the flank of NOD-scid IL2Rgamma_(mull) mice. Matrigel was added to 10⁶ cells in keratinocyte media (1:1 ratio) for each injection. Mice were monitored for tumor growth and tumors were extracted for subsequent analyses. This protocol is compliant with all ethical regulations and was approved by UCSF-Institutional Animal Care and Use Committee (IACUC).

2. Results

i. Individual Mutations are not Sufficient for Oncogenic Transformation

Multiple putative driver mutations in HNSCC have been identified by algorithms that predict the significance of recurrent mutations in large sets of sequenced tumors, the majority of which function as tumor suppressors (14, 15) (FIG. 15A). To evaluate the biological effect of 18 of the most common and significantly mutated putative driver mutations, we utilized the CRISPR/Cas9 system for targeted gene disruption of each of these genes individually in human primary foreskin keratinocytes. A mutation in at least one of these 18 genes is present in ˜93% of HNSCC cases (TCGA data), and ˜83% of these tumors show alterations in at least three of these genes (FIG. 16 ). The editing efficiency in primary keratinocytes for any individual gene ranged from 1-20% (19) (FIG. 15B). When grown in non-sorted populations, the majority of these single mutations did not provide a selective advantage over unedited cells in the same culture; only EP300^(mut) cells overtook non-edited keratinocytes with repeat passaging (FIG. 15C). To assess whether the disruption of any of these single genes is sufficient to trigger other characteristics of malignant transformation, particularly invasion, organotypic cultures with non-sorted, genome-edited primary keratinocytes were generated. In this system, primary keratinocytes are grown on the apical basement membrane of fibroblast-impregnated human dermis and form a stratified squamous epithelium, thus recapitulating the three dimensional structure of human skin or squamous mucosa (20). Among cultures of non-sorted, genome-edited keratinocytes, no single gene mutation, including EP300, was sufficient to induce lesions with characteristics of preneoplasia at the histological level as assessed by a blinded pathologist (FIG. 15D). FACS selection was then used to facilitate isolation of >90% pure cultures of edited keratinocytes (FIG. 15B). To assess whether single gene disruption was sufficient to immortalize cells, we performed serial passaging of FACS-purified, edited keratinocytes (FIG. 15E). In this case, only CDKN2A_(mut), TGFBR2_(mut), and CASP8^(mut) keratinocytes passaged beyond control, unedited or CCR5^(mut) genome-edited keratinocytes. After a few passages, TGFBR2^(mut) and CASP8^(mut) keratinocytes showed limited proliferation, while CDKN2A^(mut) cells continued to actively grow. Taken together, these data demonstrate that individual driver mutations are insufficient for the histopathological features of pre-cancerous or cancerous lesions, consistent with the previous finding of driver mutations in normal appearing stratified squamous epithelium (17, 18). Thus, it is hypothesized that combinations of mutations are likely required to induce the histopathological changes typical of pre-cancerous or cancerous lesions.

ii. Combinations of Mutations Transform Keratinocytes

TCGA data were next interrogated to identify the most common combinations of driver mutations in HPV-negative HNSCC. 90% of the cases with genomic alterations in CDKN2A also harbor mutations in TP53, suggesting that these two events may cooperate in SCC formation. Among tumors with TP53 and CDKN2A mutations, mutations in NOTCH1 are among the most common co-occurring genomic events in HNSCC and other non-viral related SCC (8, 12, 21, 22) (FIG. 15F, FIG. 17A-17D). To determine whether specific combinations of these mutations are sufficient to induce preneoplasia or cancer, mutations in CDKN2A, TP53, and NOTCH1 were introduced, both individually and in combination, in primary human keratinocytes. Keratinocytes harboring mutations in CDKN2A and TP53 (CT^(mut) cells) or CDKN2A, TP53, and NOTCH1 (CTN^(mut) cells) gained a selective advantage in vitro and were immortalized in two-dimensional culture, while keratinocytes with other combinations of these three genetic alterations ceased proliferating within a few passages (FIG. 18A). To further assess the biological effects of these mutations, populations of genome-engineered keratinocytes harboring combinations of mutations were FACS-sorted. Only CDKN2A^(mut), CT^(mut), and CTN^(mut) keratinocytes could be recovered and sequentially passaged (FIG. 18B). Sanger sequencing and TIDE analysis confirmed the expected genomic alterations in all sorted and expanded conditions (FIG. 18C). These results indicate that TP53^(mut) or TP53/NOTCH1^(mut) keratinocytes required the additional mutation of CDKN2A for survival. In a time course performed in two dimensional culture, at early time points there were no differences in cell numbers between any of the mutant conditions and control edited keratinocytes (FIG. 18D), although some differences were detected in cell-cycle distribution (FIG. 19 ). However, while normal and CDKN2A^(mut) keratinocytes exhibited contact inhibition, CT^(mut) and CTN^(mut) keratinocytes lost contact inhibition and continued to proliferate at later time points (FIG. 18D, FIG. 19 ). In order to identify alterations in the clonogenic potential of edited keratinocytes, sphere-forming assays using low-attachment plates were performed. CTN^(mut) keratinocytes showed a higher sphere-forming rate than control edited, CDKN2A^(mut), and CT^(mut) keratinocytes (FIG. 18E). These results suggest that distinct combinations of mutations permit the acquisition of a selective advantage in distinct environmental contexts.

The defining histopathological hallmark of SCC is invasion of tumor cells through the basement membrane into the underlying mesenchymal tissue. To assess whether different combinations of CDKN2A, TP53, and NOTCH1 mutations are sufficient to induce invasion, genome-edited keratinocytes were grown as epithelial layers in organotypic cultures. Dermal invasion of individual cells or small groups of cells was only detected in cultures of CTN^(mut) keratinocytes within a week of culture, confirming that the combination of these three mutations is sufficient to induce a cancer-like phenotype in human keratinocytes (FIG. 20A). CT^(mut) keratinocytes were able to form pre-neoplastic intra-epithelial lesions with enhanced proliferation, keratin pearls, and enlarged nuclei, but did not invade the basement membrane (FIG. 20A). To characterize the impact of our engineered mutations on proliferation and differentiation state in organotypic cultures, we stained for Ki67 and CD40 (ΔNp63), a marker of basal keratinocytes (FIG. 20B). CTN^(mut) keratinocytes that have invaded the dermis express both of these markers; demonstrating that invasive cells are proliferating and retain expression of basal squamous markers. After 3 weeks of continued in vitro culture CTN^(mut) keratinocytes form larger nests of invasive cells (FIG. 20C, FIG. 21 ). Further, CTN^(mut) keratinocytes injected into Nod-Scid-gamma mice were capable of tumor formation in vivo, whereas CT^(mut) cells were not. Mice injected with CTN^(mut) keratinocytes showed a poor survival rate (FIG. 20D, FIG. 22 ). Sanger sequencing confirmed the presence of the predicted mutations in CDKN2A, TP53, and NOTCH1 in tumor cells grown in vivo (FIG. 22C).

It is possible that, in the course of genome editing, additional oncogenic mutations accrue and contribute to the phenotypes we observed. In order to rule out the contribution of continued evolution and acquisition of additional genomic alterations in our edited and sorted keratinocytes, we performed capture-based next generation sequencing targeting a panel of 479 cancer-related genes and regulatory elements at a depth of 500λ. We detected no mutations or copy number alterations other than those intentionally introduced with CRISPR/Cas9 editing (FIG. 23 , Tables 6-7). In order to ensure that the transformation of keratinocytes was due to disruption of the targeted genes and not non-specific genomic instability at loci not captured in the targeted panel sequencing, we created edited keratinocytes with small guide RNAs that target the same three genes but at distinct loci and we obtained similar results as above (data not shown). In addition, this oncogenic phenotype was observed as early as 20 days after editing without repeated passages, the earliest point we had sufficient numbers of cells to assay, thus reducing the likelihood of selection of a small number of clones with additional mutations.

TABLE 8 UCSF500 Panel Sequencing Results. CDKN2A^(mut) CHROM REF ALT Func Variant TranscriptID ExonicFunc AAChange chr9 CG C exonic CDKN2A NM_000077 frameshift CDKN2A:NM_000077:exon1:c.136delC:p.R46fs p.R46fs deletion CDKN2A:NM_001195132:exon1:c.136delC:p.R46fs CDKN2A:NM_058197:exon1:c.136delC:p.R46fs CDKN2A^(mut) + TP53^(mut) CHROM REF ALT Func Variant TranscriptID ExonicFunc AAChange chr17 AGGC A exonic TP53 NM_000546 nonframeshift TP53:NM_001126115:exon2:c.270_272del:p.90_91del p.222_223del deletion TP53:NM_001126116:exon2:c.270_272del:p.90_91del TP53:NM_001126117:exon2:c.270_272del:p.90_91del TP53:NM_001276697:exon2:c.189_191del:p.63_64del TP53:NM_001276698:exon2:c.189_191del:p.63_64del TP53:NM_001276699:exon2:c.189_191del:p.63_64del TP53:NM_001126118:exon5:c.549_551del:p.183_184del TP53:NM_000546:exon6:c.666_668del:p.222_223del TP53:NM_001126112:exon6:c.666_668del:p.222_223del TP53:NM_001126113:exon6:c.666_668del:p.222_223del TP53:NM_001126114:exon6:c.666_668del:p.222_223del TP53:NM_001276695:exon6:c.549_551del:p.183_184del TP53:NM_001276696:exon6:c.549_551del:p.183_184del TP53:NM_001276760:exon6:c.549_551del:p.183_184del TP53:NM_001276761:exon6:c.549_551del:p.183_184del chr17 CCTCAGGC C exonic TP53 NM_000546 nonframeshift TP53:NM_001126115:exon2:c.268_276del:p.90_92del CC p.222_224del deletion TP53:NM_001126116:exon2:c.268_276del:p.90_92del TP53:NM_001126117:exon2:c.268_276del:p.90_92del TP53:NM_001276697:exon2:c.187_195del:p.63_65del TP53:NM_001276698:exon2:c.187_195del:p.63_65del TP53:NM_001276699:exon2:c.187_195del:p.63_65del TP53:NM_001126118:exon5:c.547_555del:p.183_185del TP53:NM_000546:exon6:c.664_672del:p.222_224del TP53:NM_001126112:exon6:c.664_672del:p.222_224del TP53:NM_001126113:exon6:c.664_672del:p.222_224del TP53:NM_001126114:exon6:c.664_672del:p.222_224del TP53:NM_001276695:exon6:c.547_555del:p.183_185del TP53:NM_001276696:exon6:c.547_555del:p.183_185del TP53:NM_001276760:exon6:c.547_555del:p.183_185del TP53:NM_001276761:exon6:c.547_555del:p.183_185del chr17 GGCGGC G exonic TP53 NM_000546 frameshift TP53:NM_001126115:exon2:c.267_271del:p.E89fs p.E221fs deletion TP53:NM_001126116:exon2:c.267_271del:p.E89fs TP53:NM_001126117:exon2:c.267_271del:p.E89fs TP53:NM_001276697:exon2:c.186_190del:p.E62fs TP53:NM_001276698:exon2:c.186_190del:p.E62fs TP53:NM_001276699:exon2:c.186_190del:p.E62fs TP53:NM_001126118:exon5:c.546_550del:p.E182fs TP53:NM_000546:exon6:c.663_667del:p.E221fs TP53:NM_001126112:exon6:c.663_667del:p.E221fs TP53:NM_001126113:exon6:c.663_667del:p.E221fs TP53:NM_001126114:exon6:c.663_667del:p.E221fs TP53:NM_001276695:exon6:c.546_550del:p.E182fs TP53:NM_001276696:exon6:c.546_550del:p.E182fs TP53:NM_001276760:exon6:c.546_550del:p.E182fs TP53:NM_001276761:exon6:c.546_550del:p.E182fs chr17 TGCAAACC T exonic TP53 NM_000546 frameshift TP53:NM_001126115:exon2:c.260_276del:p.P87fs AGACCTCA p.P219fs deletion TP53:NM_001126116:exon2:c.260_276del:p.P87fs GGCGGCTC TP53:NM_001126117:exon2:c.260_276del:p.P87fs ATAGG TP53:NM_001276697:exon2:c.179_195del:p.P60fs TP53:NM_001276698:exon2:c.179_195del:p.P60fs TP53:NM_001276699:exon2:c.179_195del:p.P60fs TP53:NM_001126118:exon5:c.539_555del:p.P180fs TP53:NM_000546:exon6:c.656_672del:p.P219fs TP53:NM_001126112:exon6:c.656_672del:p.P219fs TP53:NM_001126113:exon6:c.656_672del:p.P219fs TP53:NM_001126114:exon6:c.656_672del:p.P219fs TP53:NM_001276695:exon6:c.539_555del:p.P180fs TP53:NM_001276696:exon6:c.539_555del:p.P180fs TP53:NM_001276760:exon6:c.539_555del:p.P180fs TP53:NM_001276761:exon6:c.539_555del:p.P180fs chr17 AG A exonic TP53 NM_000546 frameshift TP53:NM_001126115:exon2:c.272delC:p.P91fs p.P223fs deletion TP53:NM_001126116:exon2:c.272delC:p.P91fs TP53:NM_001126117:exon2:c.272delC:p.P91fs TP53:NM_001276697:exon2:c.191delC:p.P64fs TP53:NM_001276698:exon2:c.191delC:p.P64fs TP53:NM_001276699:exon2:c.191delC:p.P64fs TP53:NM_001126118:exon5:c.551delC:p.P184fs TP53:NM_000546:exon6:c.668delC:p.P223fs TP53:NM_001126112:exon6:c.668delC:p.P223fs TP53:NM_001126113:exon6:c.668delC:p.P223fs TP53:NM_001126114:exon6:c.668delC:p.P223fs TP53:NM_001276695:exon6:c.551delC:p.P184fs TP53:NM_001276696:exon6:c.551delC:p.P184fs TP53:NM_001276760:exon6:c.551delC:p.P184fs TP53:NM_001276761:exon6:c.551delC:p.P184fs chr17 G GC exonic TP53 NM_000546 frameshift TP53:NM_001126115:exon2:c.271_272insG:p.P91fs p.P223fs deletion TP53:NM_001126116:exon2:c.271_272insG:p.P91fs TP53:NM_001126117:exon2:c.271_272insG:p.P91fs TP53:NM_001276697:exon2:c.190_191insG:p.P64fs TP53:NM_001276698:exon2:c.190_191insG:p.P64fs TP53:NM_001276699:exon2:c.190_191insG:p.P64fs TP53:NM_001126118:exon5:c.550_551insG:p.P184fs TP53:NM_000546:exon6:c.667_668insG:p.P223fs TP53:NM_001126112:exon6:c.667_668insG:p.P223fs TP53:NM_001126113:exon6:c.667_668insG:p.P223fs TP53:NM_001126114:exon6:c.667_668insG:p.P223fs TP53:NM_001276695:exon6:c.550_551insG:p.P184fs TP53:NM_001276696:exon6:c.550_551insG:p.P184fs TP53:NM_001276760:exon6:c.550_551insG:p.P184fs TP53:NM_001276761:exon6:c.550_551insG:p.P184fs chr17 CAG C exonic TP53 NM_000546 frameshift TP53:NM_001126115:exon2:c.272_273del:p.P91fs p.P223fs deletion TP53:NM_001126116:exon2:c.272_273del:p.P91fs TP53:NM_001126117:exon2:c.272_273del:p.P91fs TP53:NM_001276697:exon2:c.191_192del:p.P64fs TP53:NM_001276698:exon2:c.191_192del:p.P64fs TP53:NM_001276699:exon2:c.191_192del:p.P64fs TP53:NM_001126118:exon5:c.551_552del:p.P184fs TP53:NM_000546:exon6:c.668_669del:p.P223fs TP53:NM_001126112:exon6:c.668_669del:p.P223fs TP53:NM_001126113:exon6:c.668_669del:p.P223fs TP53:NM_001126114:exon6:c.668_669del:p.P223fs TP53:NM_001276695:exon6:c.551_552del:p.P184fs TP53:NM_001276696:exon6:c.551_552del:p.P184fs TP53:NM_001276760:exon6:c.551_552del:p.P184fs TP53:NM_001276761:exon6:c.551_552del:p.P184fs chr9 GACCGTA G exonic CDKN2A NM_000077 nonframeshift CDKN2A:NM_000077:exon1:c.130_135del:p.44_45del p.44_45del deletion CDKN2A:NM_001195132:exon1:c.130_135del:p.44_45del CDKN2A:NM_058197:exon1:c.130_135del:p.44_45del chr9 TCCGA T exonic CDKN2A NM_000077 frameshift CDKN2A:NM_000077:exon1:c.135_138del:p.G45fs p.G45fs deletion CDKN2A:NM_001195132:exon1:c.135_138del:p.G45fs CDKN2A:NM_058197:exon1:c.135_138del:p.G45fs chr9 A AT exonic CDKN2A NM_000077 frameshift CDKN2A:NM_000077:exon1:c.134_135insA:p.G45fs p.G45fs deletion CDKN2A:NM_001195132:exon1:c.134_135insA:p.G45fs CDKN2A:NM_058197:exon1:c.134_135insA:p.G45fs chr9 G GA exonic CDKN2A NM_000077 frameshift CDKN2A:NM_000077:exon1:c.135dupT:p.R46fs p.R46fs deletion CDKN2A:NM_001195132:exon1:c.135dupT:p.R46fs CDKN2A:NM_058197:exon1:c.135dupT:p.R46fs chr9 CG C exonic CDKN2A NM_000077 frameshift CDKN2A:NM_000077:exon1:c.136delC:p.R46fs p.R46fs deletion CDKN2A:NM_001195132:exon1:c.136delC:p.R46fs CDKN2A:NM_058197:exon1:c.136delC:p.R46fs CDKN2A^(mut) + TP53^(mut) + NOTCH1^(mut) CHROM REF ALT Func Variant TranscriptID ExonicFunc AAChange chr17 GGC G exonic TP53 NM_000546 frameshift TP53:NM_001126115:exon2:c.270_271del:p.P90fs p.P222fs deletion TP53:NM_001126116:exon2:c.270_271del:p.P90fs TP53:NM_001126117:exon2:c.270_271del:p.P90fs TP53:NM_001276697:exon2:c.189_190del:p.P63fs TP53:NM_001276698:exon2:c.189_190del:p.P63fs TP53:NM_001276699:exon2:c.189_190del:p.P63fs TP53:NM_001126118:exon5:c.549_550del:p.P183fs TP53:NM_000546:exon6:c.666_667del:p.P222fs TP53:NM_001126112:exon6:c.666_667del:p.P222fs TP53:NM_001126113:exon6:c.666_667del:p.P222fs TP53:NM_001126114:exon6:c.666_667del:p.P222fs TP53:NM_001276695:exon6:c.549_550del:p.P183fs TP53:NM_001276696:exon6:c.549_550del:p.P183fs TP53:NM_001276760:exon6:c.549_550del:p.P183fs TP53:NM_001276761:exon6:c.549_550del:p.P183fs chr17 G GT TP53 NM_000546 frameshift TP53:NM_001126115:exon2:c.271_272insA:p.P91fs p.P222fs deletion TP53:NM_001126116:exon2:c.271_272insA:p.P91fs TP53:NM_001126117:exon2:c.271_272insA:p.P91fs TP53:NM_001276697:exon2:c.190_191insA:p.P64fs TP53:NM_001276698:exon2:c.190_191insA:p.P64fs TP53:NM_001276699:exon2:c.190_191insA:p.P64fs TP53:NM_001126118:exon5:c.550_551insA:p.P184fs TP53:NM_000546:exon6:c.667_668insA:p.P223fs TP53:NM_001126112:exon6:c.667_668insA:p.P223fs TP53:NM_001126113:exon6:c.667_668insA:p.P223fs TP53:NM_001126114:exon6:c.667_668insA:p.P223fs TP53:NM_001276695:exon6:c.550_551insA:p.P184fs TP53:NM_001276696:exon6:c.550_551insA:p.P184fs TP53:NM_001276760:exon6:c.550_551insA:p.P184fs TP53:NM_001276761:exon6:c.550_551insA:p.P184fs chr9 TCCGA T exonic CDKN2A NM_000077 frameshift CDKN2A:NM_000077:exon1:c.135_138del:p.G45fs p.G45fs deletion CDKN2A:NM_001195132:exon1:c.135_138del:p.G45fs CDKN2A:NM_058197:exon1:c.135_138del:p.G45fs chr9 TAC T exonic NOTCH1 NM_017617 frameshift NOTCH1:NM_017617:exon6:c.875_876del:p.C292fs p.C292fs deletion chr9 G GT exonic NOTCH1 NM_017617 frameshift NOTCH1:NM_017617:exon6:c.877dupA:p.T293fs p.T293fs deletion

Previous studies have highlighted the similarities between SCC arising from different anatomical sites (14). To test the generalizability of our findings in another tissue of origin, we induced mutations in TP53, CDKN2A, and NOTCH1 in primary human tonsil keratinocytes and observed a similar invasive phenotype as seen in foreskin keratinocytes (FIG. 24 ). Based on similar mutation patterns detected in different non-viral SCC tumor types, this mutational pathway is likely operant across distinct tissues of origin. Thus, mutations in TP53 and CDKN2A are sufficient to immortalize but not transform keratinocytes, and the addition of NOTCH1 mutation is sufficient for the progression to cancer.

iii. Mechanisms of Keratinocyte Transformation

In order to understand how the transcriptome is reprogrammed in response to the acquisition of each mutation, we performed whole-genome RNA sequencing (RNA-seq) analysis on pure populations of genome-engineered primary human keratinocytes harboring combinations of mutations in TP53, CDKN2A, and NOTCH1. Unsupervised clustering demonstrated that CTN^(mut) keratinocytes are the most transcriptomically distinct in comparison to CT^(mut), CDKN2A_(mut), and unedited keratinocytes (FIG. 25A). Mutation in CDKN2A alone resulted in significant changes in 1,051 genes (adjusted p-value <0.05, at least twofold), including an enrichment of genes promoting cell cycle progression and a slight downregulation of genes involved in keratinocyte differentiation (FIG. 25B). The additional mutation in TP53 modified the expression of 892 genes (compared to CDKN2A^(mut) keratinocytes, adjusted p-value <0.05, at least twofold), two-thirds of which showed downregulation. GO term analysis showed a stronger activation of genes promoting the cell cycle, a decline of pro-apoptotic factors (BAX) and a downregulation of keratinocyte differentiation markers (IVL, FLG). These findings suggest that the combination of mutations in CDKN2A and TP53 is sufficient to force keratinocytes into a basal-like state. The additional mutation in NOTCH1 resulted in changes in expression of 1,020 genes (compared to CT^(mut) keratinocytes, adjusted P value <0.05, at least twofold). GO term analysis of this set of genes showed the upregulation of anti-apoptotic factors and basal-keratinocyte markers (KRT14), and a shift in the expression of adhesion molecules (ITGA1, ITGA6, ICAM1). Finally, NOTCH1 mutation in this background induces an EMT-like program that includes the downregulation of E-cadherin (CDH1) and activation of N-cadherin (CDH2) (FIG. 25B and FIG. 25C, FIG. 26 , Table 8). These data, along with our organotypic cultures and in vivo data, indicate that combined mutations in CDKN2A, TP53, and NOTCH1 are sufficient to transform human primary keratinocytes into cancer and is accompanied by an induction of cell-cycle progression, a block of keratinocyte differentiation, and induction of an EMT-like phenotype.

TABLE 8 Results of Differential Gene Expression Analysis. baseMean log2FoldChange lfcSE stat pvalue padj hgnc_symbol 193.2552834 5.455822457 0.275521445 19.80180695 2.87E-87 2.14E-84 CXCL5 119.5534697 -5.437311242 0.307263725 -17.69591008 4.51E-70 2.17E-67 HLA-DQB1 232.6366223 5.144915519 0.2511104 20.48865964 2.72E-93 2.47E-90 MMP10 317.9092607 5.104955547 0.211758096 24.10748698 2.09E-128 8.55E-125 NOS1 326.8426926 4.981751032 0.210310031 23.68765298 4.83E-124 1.32E-120 SOX11 170.2869601 4.293982989 0.232443098 18.47326519 3.39E-76 2.06E-73 POSTN 85.9456744 -4.244225457 0.280903645 -15.10918611 1.41E-51 3.25E-49 CTSH 205.5016868 4.144116058 0.206338861 20.08403086 1.02E-89 7.94E-87 MMP9 45.05689572 4.052548086 0.335890761 12.06507758 1.62E-33 1.58E-31 PRSS21 65.78797665 4.033267838 0.303225937 13.30119674 2.28E-40 3.11E-38 COX20P1 75.06419412 3.980568523 0.288231722 13.81030685 2.21E-43 3.45E-41 SLC10A6 94.4419079 -3.807459634 0.254898578 -14.93715523 1.89E-50 4.18E-48 LINC01116 39.8848753 3.708290877 0.333774268 11.11017604 1.12E-28 8.41E-27 IRX1 68.2966123 -3.662958176 0.279872441 -13.08795595 3.86E-39 5.02E-37 CYBA 119.3423637 3.661589119 0.234794175 15.59488907 7.89E-55 2.08E-52 STEAP1B 121.6866391 -3.628795263 0.225262189 -16.10920711 2.20E-58 6.55E-56 FOXA2 34.47239949 3.597215399 0.341582928 10.53101636 6.22E-26 3.96E-24 CCDC152 31.17675173 3.575869922 0.346332214 10.32497057 5.43E-25 3.27E-23 ESM1 32.3719363 3.557448277 0.341315331 10.42276145 1.95E-25 1.18E-23 FAM155B 44.13309552 3.548420657 0.326538509 10.86677546 1.66E-27 1.16E-25 LGALS7 51.44890307 3.507282521 0.303400148 11.55992358 6.58E-31 5.53E-29 LINC00960 28.72141455 3.470609939 0.348509472 9.958437923 2.32E-23 1.29E-21 LEF1 65.35262506 -3.377708064 0.274067456 -12.32436758 6.70E-35 6.94E-33 SLFN11 562.5274469 3.361952194 0.146405427 22.96330314 1.09E-116 2.22E-113 MMP1 150.2488859 3.349403495 0.207172079 16.16725337 8.58E-59 2.70E-56 TNFSF15 138.2038405 -3.30638207 0.204586075 -16.16132508 9.45E-59 2.92E-56 CLDN11 195.8720888 3.305217735 0.189296702 17.46051413 2.86E-68 1.20E-65 ERAP2 49.26922676 3.270132562 0.305611048 10.70030871 1.01E-26 6.78E-25 TUBBP5 149.907814 -3.172460921 0.197081933 -16.09716764 2.67E-58 7.81E-56 TMEM204 136.0541136 -3.162250264 0.204822872 -15.43895093 8.95E-54 2.19E-51 CSF3 20.59896487 -3.145677503 0.355970379 -8.836908037 9.84E-19 4.02E-17 PSG4 70.68601126 3.080107509 0.261329959 11.78627784 4.59E-32 4.05E-30 FGFR1 20.71712319 -3.066028997 0.353603122 -8.670819931 4.29E-18 1.71E-16 MTND4P24 22.61624969 3.055359717 0.353034417 8.654566155 4.95E-18 1.96E-16 EEF1GPI 61.9830611 3.004836719 0.269773633 11.13836326 8.16E-29 6.22E-27 CPPED1 59.06606914 -2.993291106 0.270674916 -11.05862024 1.99E-28 1.47E-26 PNMA8A 18.5786669 -2.96567328 0.359531553 -8.248714902 1.60E-16 5.64E-15 NA 20.21113501 2.942485776 0.354782711 8.293768784 1.10E-16 3.90E-15 TTN-AS1 35.93213777 2.939208607 0.315203281 9.32480335 1.11E-20 5.28E-19 NDN 18.87734182 2.929511337 0.358894666 8.162593692 3.28E-16 1.13E-14 ZNF280B 37.02685519 2.91968605 0.320566772 9.10788737 8.40E-20 3.69E-18 IGFL3 74.83313552 2.871364637 0.248516563 11.55401717 7.04E-31 5.86E-29 RASGEF1A 20.2206575 2.845879851 0.352738767 8.067953166 7.15E-16 2.39E-14 FAM180A 40.55482875 -2.820908066 0.303245068 -9.302403788 1.37E-20 6.50E-19 HOXB4 73.65560894 2.802847632 0.253876137 11.04021696 2.44E-28 1.80E-26 SCN8A 18.40722129 2.801676489 0.357467248 7.83757535 4.59E-15 1.42E-13 APCDD1 274.9302689 -2.796172714 0.154717754 -18.0727333 5.23E-73 2.68E-70 CNTN1 63.0177826 -2.79026694 0.25927966 -10.76161137 5.22E-27 3.55E-25 TRHDE-AS1 16.7332851 2.789531398 0.360993747 7.727367636 1.10E-14 3.28E-13 LINC01835 30.66733182 -2.743168831 0.322579598 -8.503850975 1.83E-17 7.00E-16 FAM198B- AS1 82.48211968 -2.741884315 0.237801692 -11.53012956 9.30E-31 7.58E-29 NTSR1 256.605606 2.739428025 0.171963116 15.93032325 3.90E-57 1.10E-54 KRT75 133.7976973 -2.736785133 0.20000764 -13.68340294 1.28E-42 1.90E-40 PINLYP 2029.067072 2.734202558 0.09255845 29.54028039 8.75E-192 1.43E-187 SPRR2D 434.5634113 -2.733645321 0.128691907 -21.24178105 3.93E-100 4.95E-97 MME 19.487628 -2.723311855 0.354836415 -7.674837593 1.66E-14 4.88E-13 TLR2 605.850793 -2.713118168 0.114532868 -23.68855523 4.73E-124 1.32E-120 BST2 29.96136566 -2.701553842 0.324083527 -8.335980145 7.69E-17 2.77E-15 TYWIB 31.49047422 2.683267362 0.320801889 8.364250512 6.05E-17 2.20E-15 LINC01322 15.19997056 2.678500627 0.362494161 7.389086274 1.48E-13 4.03E-12 NAP1L2 16.7365735 -2.666280869 0.357996209 -7.447790789 9.49E-14 2.63E-12 NA 105.9101021 2.635387367 0.216040881 12.19855871 3.16E-34 3.24E-32 SPINK6 14.85509634 2.631828016 0.36321616 7.245900119 4.30E-13 1.12E-11 TNS1 47.41360713 2.626300022 0.284756293 9.222974478 2.89E-20 1.33E-18 COL5A1 17.88633829 -2.618691439 0.356440584 -7.346782488 2.03E-13 5.48E-12 DPYSL4 25.82810001 -2.610143168 0.333836616 -7.818624582 5.34E-15 1.64E-13 PI15 37.67742299 2.582483611 0.308283094 8.376987442 5.43E-17 1.99E-15 COL11A1 48.59944078 2.581612979 0.282001968 9.154592051 5.46E-20 2.44E-18 APBA2 77.08575695 -2.565440724 0.23164897 -11.07469082 1.66E-28 1.23E-26 CFH 100.9275676 2.562438196 0.217764584 11.76701075 5.77E-32 5.03E-30 CAPNS2 13.75352406 2.56164031 0.363903995 7.039330002 1.93E-12 4.77E-11 KIAA1324L 27.62244064 2.560487574 0.326327066 7.846384327 4.28E-15 1.33E-13 TSPYL5 112.6127226 2.552388182 0.210573486 12.12112803 8.16E-34 8.10E-32 SCG5 233.2355295 2.551500464 0.153179665 16.65691375 2.70E-62 9.60E-60 RBP1 64.38105596 -2.545067186 0.248291816 -10.25030637 1.18E-24 7.03E-23 TREM2 46.75753988 -2.541791352 0.291201019 -8.728648561 2.58E-18 1.04E-16 LOXL2 13.65974313 2.524651752 0.364443876 6.9274089 4.29E-12 1.02E-10 DACT2 66.43295129 -2.512669783 0.246651292 -10.18713406 2.26E-24 1.33E-22 HCLS1 13.8386727 -2.500794119 0.363323811 -6.88309998 5.86E-12 1.37E-10 NA 96.48423891 2.496211243 0.220216135 11.33527863 8.78E-30 6.88E-28 CXCL3 80.81679305 -2.490941704 0.238628397 -10.43858037 1.65E-25 1.02E-23 HOXB7 20.36911674 2.479685789 0.346702098 7.152208783 8.54E-13 2.17E-11 PCDHGA12 48.72701937 -2.474322973 0.269364452 -9.185781386 4.09E-20 1.85E-18 ORC1 14.04194241 2.465904903 0.362897158 6.795051567 1.08E-11 2.46E-10 PEG3 410.1386314 -2.461857711 0.127342184 -19.33261724 2.86E-83 1.95E-80 PLIN2 208.1265004 2.461031345 0.159113903 15.46710438 5.79E-54 1.44E-51 ABCG1 24.82019172 2.45430812 0.340159934 7.21515932 5.39E-13 1.39E-11 TMPRSS11B 181.160236 2.452772954 0.17630328 13.91223664 5.34E-44 8.57E-42 ULBP1 49.99665332 -2.440931154 0.268614017 -9.087132471 1.02E-19 4.44E-18 LAMA1 73.51892017 2.440408847 0.243088458 10.03918027 1.03E-23 5.79E-22 CLIP3 23.24301771 -2.428569394 0.334008474 -7.270981383 3.57E-13 9.38E-12 NA 293.6154369 2.424161871 0.163295286 14.84526547 7.47E-50 1.57E-47 KRT31 41.27560508 -2.417675258 0.285533607 -8.467217859 2.51E-17 9.44E-16 TRHDE 179.4934952 2.415938056 0.171170833 14.11419231 3.11E-45 5.41E-43 KRT34 246.7120125 2.406882043 0.153474872 15.68258054 1.99E-55 5.43E-53 KRTAP2-3 264.5492841 -2.387294896 0.155524872 -15.34992355 3.55E-53 8.54E-51 DTL 14.45865876 2.385130954 0.361304793 6.601437349 4.07E-11 8.54E-10 VLDLR-AS1 31.03935143 -2.366616631 0.316325985 -7.481575149 7.34E-14 2.06E-12 TWIST1 136.8287767 2.364297739 0.191059492 12.37466779 3.58E-35 3.76E-33 NA 40.84692217 2.363692423 0.291523353 8.108072308 5.14E-16 1.75E-14 NEGR1 11.62133441 -2.346640227 0.366337783 -6.405673492 1.50E-10 2.92E-09 MTCYBP18 715.1936794 2.345087572 0.116428945 20.14179178 3.18E-90 2.60E-87 SPRR2A 11.27150601 2.302071121 0.366167499 6.28693461 3.24E-10 6.12E-09 NA 180.4522163 2.294152489 0.17382784 13.1978427 9.03E-40 1.19E-37 ADM2 79.85794551 2.293746657 0.23897686 9.598195634 8.14E-22 4.14E-20 MEIS3 19.81722945 2.292773692 0.344577626 6.653866992 2.85E-11 6.08E-10 SYNGR3 12.73796574 -2.289669182 0.36391529 -6.291764175 3.14E-10 5.97E-09 HOXB3 22.72718407 2.281862186 0.334994498 6.811640783 9.65E-12 2.21E-10 HLA-G 127.8863812 2.273236999 0.341663017 6.653447655 2.86E-11 6.09E-10 SPRR2G 412.8061047 -2.270491722 0.122569168 -18.52416688 1.32E-76 8.64E-74 ELP5 51.99990918 2.267050982 0.267387623 8.478518768 2.28E-17 8.63E-16 BEND7 288.5597217 -2.259611424 0.142490381 -15.85799273 1.24E-56 3.44E-54 MCM10 13.20253984 -2.258151995 0.3621325 -6.235706525 4.50E-10 8.34E-09 HERC2P3 95.89548054 2.252211295 0.220162942 10.22974744 1.46E-24 8.66E-23 PYGO1 73.80823575 -2.248081273 0.232306178 -9.677234123 3.77E-22 1.96E-20 CCNE2 10.73462634 2.23561157 0.366470877 6.100379901 1.06E-09 1.89E-08 SLC27A6 12.3856895 2.218865278 0.363543712 6.103434624 1.04E-09 1.86E-08 BRSK1 288.986101 -2.218303356 0.166446865 -13.32739644 1.60E-40 2.21E-38 SPRR3 427.9473485 -2.211348475 0.122105868 -18.11009171 2.65E-73 1.40E-70 FAM111B 217.2075559 2.205812354 0.185253015 11.90702542 1.09E-32 9.85E-31 PRSS3 31.67238824 2.203978647 0.306599335 7.188465197 6.55E-13 1.68E-11 RTN4RL1 39.6965331 -2.197407295 0.287320341 -7.647935005 2.04E-14 5.95E-13 SAA2 23.17833281 -2.194676146 0.335992105 -6.531927734 6.49E-11 1.32E-09 CX3CL1 26.42856356 -2.187032996 0.322670706 -6.777909974 1.22E-11 2.74E-10 SLFN13 202.4481448 2.180193528 0.160378078 13.59408688 4.34E-42 6.35E-40 IRX3 25.48879009 2.174395973 0.330767824 6.573783226 4.91E-11 1.01E-09 ICA1 179.1168414 -2.151952109 0.161188889 -13.35049901 1.18E-40 1.65E-38 CSPG4 26.51078412 -2.147327677 0.328035123 -6.546029765 5.91E-11 1.21E-09 DISP2 193.4701371 -2.146103148 0.171864008 -12.4872169 8.77E-36 9.70E-34 MTND4P12 225.0421525 -2.14107858 0.15631532 -13.69717687 1.06E-42 1.59E-40 EPSTI1 44.65189814 2.137580686 0.273693692 7.8101204 5.71E-15 1.75E-13 ZFP57 25.59630038 2.13704612 0.330965567 6.457004392 1.07E-10 2.13E-09 BEX5 80.64154665 -2.133347352 0.232659656 -9.169390986 4.76E-20 2.14E-18 NRGN 128.1639336 -2.133153944 0.184877469 -11.53820395 8.47E-31 6.94E-29 SYK 35.42764864 -2.133019063 0.293985105 -7.255534459 4.00E-13 1.05E-11 SLC15A3 636.905113 -2.129054516 0.105308615 -20.21728731 6.90E-91 5.95E-88 PCLAF 711.2025111 -2.118304009 0.10306493 -20.55310189 7.22E-94 6.96E-91 NCAPG 10.52281001 2.117947661 0.366079052 5.785492647 7.23E-09 1.19E-07 C6orf223 20.48244389 2.112016072 0.340553 6.201725052 5.58E-10 1.03E-08 TBX1 56.67442884 -2.109680906 0.250657984 -8.416571733 3.88E-17 1.43E-15 FAM198B 342.8251762 2.105341552 0.127668871 16.49064131 4.28E-61 1.46E-58 ALDH1L2 14.4837212 -2.103330547 0.357987185 -5.875435314 4.22E-09 7.08E-08 NLRP3 19.42518485 -2.100790598 0.340585514 -6.16817366 6.91E-10 1.26E-08 ANKUB1 11.04067014 -2.087165175 0.365843244 -5.705080552 1.16E-08 1.84E-07 CASP14 12.38829908 2.087041578 0.36336423 5.743662712 9.27E-09 1.49E-07 ANKRD18CP 1484.383123 2.083809799 0.093092535 22.38428464 5.60E-111 1.02E-107 PTGS2 85.81359762 2.081296172 0.222852648 9.339337855 9.69E-21 4.62E-19 LY6D 27.08055104 -2.075914395 0.319607089 -6.495207608 8.29E-11 1.67E-09 RNY1 33.48038568 -2.072708984 0.306861072 -6.754551727 1.43E-11 3.18E-10 METTL7A 13.34851317 2.068195494 0.361631184 5.719073975 1.07E-08 1.71E-07 CA5BP1 37.72604575 2.062812159 0.287962921 7.163464494 7.87E-13 2.00E-11 PTPRS 13.83290791 -2.05589431 0.35853525 -5.734148348 9.80E-09 1.57E-07 MYBPHL 1095.114242 2.046349103 0.257742865 7.939498567 2.03E-15 6.55E-14 KRT16P6 10.40339263 -2.042791655 0.366070168 -5.58032812 2.40E-08 3.63E-07 AKNAD1 26.14922077 -2.041899593 0.319138323 -6.398164824 1.57E-10 3.06E-09 PTMAP9 184.8756643 -2.038474815 0.158028259 -12.89943224 4.53E-38 5.63E-36 ELAVL2 16.79308946 2.036262138 0.352172388 5.782003945 7.38E-09 1.21E-07 ADAM23 69.66970036 -2.034125588 0.236240922 -8.610386276 7.28E-18 2.85E-16 ZNF367 265.3695946 -2.033113631 0.136433733 -14.90183978 3.21E-50 6.82E-48 CDT1 10.85696899 2.032505458 0.365608103 5.559246199 2.71E-08 4.07E-07 CCDC170 94.66243915 2.032455792 0.214929209 9.456396385 3.19E-21 1.56E-19 SV2A 80.27223865 -2.027979957 0.223592397 -9.06998622 1.19E-19 5.17E-18 ERCC6L 75.23335273 -2.022451081 0.22470974 -9.00028223 2.25E-19 9.58E-18 PTGS1 141.7935608 2.021300895 0.339632089 5.951442628 2.66E-09 4.57E-08 CDSN 60.51209378 2.018977471 0.254399369 7.93625187 2.08E-15 6.69E-14 SLC9A3-AS1 194.0344515 2.016811912 0.160269717 12.58386146 2.59E-36 2.95E-34 CWH43 82.38987592 2.015669317 0.218338258 9.231864962 2.66E-20 1.24E-18 PLEKHG4 10.66648343 2.013504988 0.365618509 5.507119959 3.65E-08 5.40E-07 LINC00958 66.04695737 -2.002933895 0.244702765 -8.185170662 2.72E-16 9.44E-15 C17orf97 165.9991954 2.002252282 0.164898316 12.14234523 6.30E-34 6.29E-32 CXCL1 156.1368395 -1.999614636 0.168554937 -11.86328133 1.84E-32 1.64E-30 GDF15 8.900168294 1.993533318 0.366304517 5.442284287 5.26E-08 7.63E-07 PSPHP1 66.96724802 1.992932724 0.239017756 8.338011198 7.56E-17 2.73E-15 RORA 48.5247778 -1.992407638 0.27938349 -7.131443735 9.93E-13 2.50E-11 PRR9 253.3546581 -1.990842971 0.142628932 -13.9581987 2.80E-44 4.64E-42 CDC6 28.00322572 1.98441396 0.31120878 6.376471645 1.81E-10 3.51E-09 ASPHD1 199.4477413 1.983964057 0.169128092 11.73054126 8.89E-32 7.62E-30 ASS1 163.8429283 -1.979580617 0.165909673 -11.93167692 8.09E-33 7.45E-31 CDCA5 1065.197846 -1.975099139 0.089571255 -22.05059136 9.43E-108 1.54E-104 ANLN 931.046002 -1.959881892 0.093885653 -20.87520124 9.00E-97 9.83E-94 NEFL 31.48860984 1.959679238 0.306770326 6.388099088 1.68E-10 3.26E-09 EMX20S 9.063336688 1.951716982 0.36665001 5.323106308 1.02E-07 1.42E-06 NA 24.42742652 -1.949492222 0.328517101 -5.934218392 2.95E-09 5.05E-08 CCDC144B 377.588117 1.94945331 0.147457425 13.22044864 6.69E-40 8.91E-38 KRT6C 154.3165641 -1.949452443 0.185991882 -10.48138459 1.05E-25 6.58E-24 FOS 181.6961861 -1.945839017 0.173393674 -11.2220877 3.18E-29 2.44E-27 ESCO2 11.7038359 -1.942363097 0.364986036 -5.321746327 1.03E-07 1.42E-06 DEPP1 1103.984753 -1.940610866 0.094167881 -20.60799131 2.33E-94 2.38E-91 SUGCT 83.03286734 -1.939582637 0.213055101 -9.103666742 8.73E-20 3.83E-18 CCNJL 50.57339379 -1.935126945 0.270047485 -7.165876557 7.73E-13 1.97E-11 VNN1 44.97779595 1.928912881 0.276955444 6.964704691 3.29E-12 7.93E-11 PAPPA2 65.52225402 -1.927655993 0.231480901 -8.327494753 8.26E-17 2.96E-15 EXO1 235.5065066 -1.927165538 0.154547106 -12.46976141 1.09E-35 1.20E-33 ASF1B 185.1118597 -1.924872803 0.163322858 -11.78569142 4.63E-32 4.05E-30 FAM83D 9.414814331 1.922342762 0.366629409 5.243285774 1.58E-07 2.12E-06 NA 24.41812926 -1.921776217 0.324905507 -5.914877318 3.32E-09 5.66E-08 SP140 14.85408028 1.921018519 0.357406937 5.374877541 7.66E-08 1.09E-06 TDRD12 172.4035624 -1.920416909 0.173003069 -11.10047888 1.25E-28 9.33E-27 GINS1 338.0499431 -1.919061781 0.137604187 -13.94624559 3.32E-44 5.43E-42 HJURP 193.149451 1.916093155 0.182454888 10.50173651 8.48E-26 5.34E-24 SDCBP2 25.90263752 -1.911119595 0.313346722 -6.09905725 1.07E-09 1.90E-08 PLCL2 768.3692384 -1.907297756 0.104774804 -18.20378268 4.82E-74 2.63E-71 CDC20 8.774476701 1.904611765 0.366625323 5.194981483 2.05E-07 2.71E-06 ZNF736P9Y 164.6713754 -1.903636314 0.161985414 -11.75189954 6.90E-32 5.99E-30 NCAPH 143.5156817 -1.902780449 0.173532553 -10.964977 5.63E-28 4.06E-26 ZFP42 8.934247452 1.899965006 0.366595881 5.182723281 2.19E-07 2.88E-06 PLBD1 136.3996527 -1.899869328 0.176983595 -10.73471997 6.99E-27 4.71E-25 GINS2 95.25373554 -1.898605987 0.203252067 -9.341139876 9.53E-21 4.55E-19 HSD17B2 76.36736733 -1.897590271 0.223331254 -8.496751946 1.95E-17 7.43E-16 SPC24 430.8431599 -1.895126472 0.114504647 -16.55065123 1.58E-61 5.52E-59 RRM2 339.5493713 -1.890739196 0.132233015 -14.29854109 2.23E-46 3.94E-44 P3H2 74.20684715 -1.889697451 0.230743016 -8.189619276 2.62E-16 9.12E-15 CENPM 56.94277123 -1.889423126 0.24530085 -7.702472803 1.33E-14 3.96E-13 SCNNIA 99.24366529 1.882013921 0.208962964 9.006447288 2.13E-19 9.13E-18 LURAP1L- AS1 514.5206285 -1.88003596 0.111554492 -16.85307266 9.96E-64 3.89E-61 CLSPN 100.6868883 1.879260023 0.217904224 8.6242478 6.45E-18 2.54E-16 EMX2 142.8285935 1.871287362 0.176878053 10.57953393 3.71E-26 2.38E-24 SYT7 38.96160171 -1.868392024 0.306257588 -6.100720753 1.06E-09 1.88E-08 KIF18B 210.5769866 -1.863186718 0.153208175 -12.16114429 5.01E-34 5.03E-32 SHCBP1 42.92831513 1.862599661 0.275499114 6.760819054 1.37E-11 3.06E-10 NKAPL 247.8056199 1.859905624 0.155510337 11.9600128 5.76E-33 5.42E-31 SEZ6L2 131.3775475 -1.858062876 0.185057618 -10.04045601 1.01E-23 5.74E-22 CDCA3 14.23054545 -1.856101118 0.355290052 -5.224185439 1.75E-07 2.34E-06 BDNF 1768.72627 -1.849394031 0.078130543 -23.67056419 7.25E-124 1.70E-120 PRELID1 11.44280892 1.847381194 0.364574278 5.067228554 4.04E-07 5.08E-06 NA 39.82877043 1.846596149 0.291078917 6.343970794 2.24E-10 4.32E-09 DUSP8 117.320078 -1.842115492 0.18620591 -9.892894888 4.47E-23 2.44E-21 FOXM1 66.7406101 -1.839713104 0.229640372 -8.011279073 1.14E-15 3.74E-14 APOL1 242.0767963 -1.829614835 0.151134376 -12.10588142 9.83E-34 9.70E-32 LMNB1 340.3973373 -1.829565881 0.124562329 -14.68795494 7.70E-49 1.56E-46 CCNA2 245.0055496 -1.823088029 0.146275815 -12.4633592 1.18E-35 1.29E-33 MYBL2 48.73892293 -1.81440256 0.288559819 -6.287786599 3.22E-10 6.09E-09 HIST1H4H 600.7276182 -1.814028339 0.10335504 -17.55142598 5.80E-69 2.72E-66 CDK1 1504.818078 -1.81375784 0.084308993 -21.51321902 1.17E-102 1.74E-99 LCP1 3615.172019 -1.805539892 0.073501747 -24.56458464 3.02E-133 1.65E-129 TOP2A 18.40398949 1.803243465 0.338585282 5.325817637 1.00E-07 1.40E-06 LY96 28.65606134 -1.802871546 0.305041175 -5.910256361 3.42E-09 5.79E-08 MCHR1 10.54527554 1.798816699 0.365136189 4.926426778 8.37E-07 1.00E-05 NA 9.982730924 -1.798368886 0.365687014 -4.917781644 8.75E-07 1.04E-05 SERPINA1 15.04824786 -1.796433467 0.351449499 -5.111498158 3.20E-07 4.09E-06 HIST1H2AH 75.3017071 1.793665922 0.223232181 8.034979156 9.36E-16 3.10E-14 DNAJA4 504.2376992 -1.789033567 0.109124348 -16.39444904 2.10E-60 6.87E-58 BIRC5 3156.829309 -1.788086201 0.071601748 -24.97266132 1.21E-137 9.93E-134 KRT7 177.6825961 -1.785929712 0.160619393 -11.11901673 1.01E-28 7.69E-27 KITLG 11.40171073 1.785765576 0.363616932 4.911117769 9.06E-07 1.08E-05 VWDE 10.02253758 -1.783292027 0.366026393 -4.87203126 1.10E-06 1.29E-05 NA 1679.648502 -1.77051668 0.095792087 -18.48291166 2.83E-76 1.79E-73 CENPF 96.28063492 -1.768245425 0.201775076 -8.763448199 1.89E-18 7.70E-17 CDC45 171.0164223 1.766585128 0.202470277 8.725157846 2.66E-18 1.07E-16 CNFN 62.96491811 1.764150355 0.241141376 7.315834336 2.56E-13 6.81E-12 CTH 81.77023419 -1.762943125 0.212221719 -8.307081543 9.81E-17 3.50E-15 GINS4 8.222208269 -1.762637055 0.366523857 -4.809065004 1.52E-06 1.72E-05 RNU4-2 219.7537201 -1.755986827 0.148698774 -11.80902025 3.51E-32 3.11E-30 RAD51AP1 408.5829967 -1.754329474 0.134586432 -13.0349653 7.74E-39 9.98E-37 PLK1 1539.617129 -1.753310348 0.095218641 -18.41352005 1.02E-75 5.99E-73 MKI67 327.2182625 -1.753261154 0.125946112 -13.92072475 4.74E-44 7.69E-42 SNHG17 78.65219902 -1.751037358 0.216869719 -8.074144081 6.80E-16 2.27E-14 SPC25 27.8046681 1.75036424 0.306197393 5.716457032 1.09E-08 1.73E-07 ZPLD1 112.0155154 1.742008882 0.187671381 9.282229804 1.66E-20 7.83E-19 HOMER2 6.859113336 1.73856748 0.363803227 4.778867664 1.76E-06 1.99E-05 COL4A4 12.27708444 1.738034251 0.359788025 4.830717346 1.36E-06 1.57E-05 MEG3 233.7200874 -1.735574182 0.152440123 -11.38528455 4.95E-30 3.92E-28 PBK 25.91878693 1.734180935 0.319992375 5.419444556 5.98E-08 8.58E-07 PPP2R2C 773.7540942 -1.734177042 0.099599473 -17.41150818 6.75E-68 2.76E-65 HELLS 8.65356821 -1.732325129 0.366629491 -4.725002143 2.30E-06 2.54E-05 HOXB5 91.84701788 1.73009621 0.21036844 8.224124335 1.97E-16 6.90E-15 SMIM22 21.00011882 1.725556217 0.334059933 5.165409092 2.40E-07 3.14E-06 PKN1 75.60006131 1.722892715 0.221368979 7.782900417 7.09E-15 2.15E-13 ATP9A 192.0767016 -1.722718104 0.157589865 -10.93165544 8.14E-28 5.85E-26 FBXO5 286.8955385 -1.717099819 0.133078148 -12.90294342 4.33E-38 5.42E-36 NDC80 74.56351907 -1.71560459 0.230022714 -7.458413812 8.76E-14 2.44E-12 E2F1 175.1297453 -1.71363848 0.1641698 -10.43820772 1.66E-25 1.02E-23 GTSE1 7.167112884 -1.713524047 0.365050161 -4.693941349 2.68E-06 2.91E-05 FYB1 12.91488155 -1.705411586 0.366197553 -4.657080783 3.21E-06 3.44E-05 LGALS7B 138.1198568 -1.705160191 0.207439083 -8.220052691 2.03E-16 7.12E-15 HIST1H1B 415.3741229 -1.703739718 0.121873689 -13.97955314 2.08E-44 3.47E-42 CKAP2L 39.33083316 1.70140069 0.27879602 6.102672099 1.04E-09 1.86E-08 HAS2 14.06830212 1.698813262 0.352946912 4.813226022 1.49E-06 1.69E-05 FOXP2 243.634594 -1.697899741 0.146261325 -11.60867192 3.72E-31 3.14E-29 MAD2L1 151.9144235 1.697628033 0.168198125 10.09302591 5.93E-24 3.41E-22 FUOM 9.474307334 -1.695775682 0.366490563 -4.627065075 3.71E-06 3.91E-05 NA 51.22477369 -1.693492093 0.248415552 -6.817174203 9.28E-12 2.13E-10 NHLH2 35.11079242 -1.690232214 0.291371438 -5.800953677 6.59E-09 1.09E-07 KCNS3 158.9901536 -1.690165534 0.161940034 -10.43698392 1.68E-25 1.03E-23 SGO1 17.94383308 1.689187027 0.339579014 4.974356359 6.55E-07 7.96E-06 TRPV4 375.8857964 -1.688793827 0.124625621 -13.55093615 7.82E-42 1.13E-39 BRCA2 21.27440492 -1.687557038 0.327812297 -5.147936955 2.63E-07 3.43E-06 HIST1H1A 48.18149313 -1.685190155 0.259631952 -6.490688626 8.54E-11 1.72E-09 IL7R 49.02715331 1.68479043 0.257620488 6.539815392 6.16E-11 1.26E-09 DIRC3 347.0378107 -1.684019028 0.122917767 -13.70037109 1.01E-42 1.53E-40 UBE2C 6.587130911 1.683901558 0.36279606 4.641454922 3.46E-06 3.67E-05 JAM3 31.97689246 1.680322483 0.29260527 5.742625491 9.32E-09 1.50E-07 EPGN 13.4447931 1.68015369 0.356526256 4.712566495 2.45E-06 2.68E-05 BAIAP2L2 216.5579165 -1.680046207 0.155930503 -10.77432689 4.55E-27 3.12E-25 DEPDC1 735.1356943 -1.679923365 0.095899937 -17.51746065 1.05E-68 4.80E-66 MCM7 258.339914 -1.678239452 0.135030164 -12.42862632 1.83E-35 1.96E-33 LIG1 1120.299638 -1.6771485 0.085602401 -19.59230669 1.80E-85 1.28E-82 IFITM2 199.4635033 -1.673715587 0.173051519 -9.671776317 3.97E-22 2.06E-20 KIFC1 25.23602888 1.667529917 0.322343452 5.173146546 2.30E-07 3.02E-06 RAB6B 24.58774715 1.666482189 0.323145702 5.157061283 2.51E-07 3.27E-06 DSG1 16.65249259 -1.660929241 0.347120933 -4.784872017 1.71E-06 1.93E-05 HIST1H2BF 24.18580892 1.653431659 0.32312796 5.116956322 3.11E-07 3.99E-06 SHC2 257.2306367 -1.652410593 0.157948433 -10.4617093 1.29E-25 8.04E-24 ATAD5 34.88957264 -1.650145645 0.29023223 -5.685604397 1.30E-08 2.05E-07 SCN2A 128.4388137 -1.64899741 0.184725094 -8.926764501 4.39E-19 1.83E-17 KIF20A 8.007359875 -1.646420684 0.366623686 -4.490764631 7.10E-06 7.10E-05 RNY4 2182.56758 -1.644388446 0.101525414 -16.19681598 5.31E-59 1.71E-56 RPL22L1 300.0836708 -1.643618309 0.127668513 -12.87410864 6.30E-38 7.70E-36 PKMYT1 367.6372256 1.642373219 0.267015998 6.150842009 7.71E-10 1.40E-08 SPRR2E 665.5254046 -1.642170171 0.112032019 -14.65804313 1.20E-48 2.39E-46 TMEM97 7.190963839 1.642105241 0.364877516 4.500428693 6.78E-06 6.83E-05 HOXC12 6.272949121 1.641957626 0.36200236 4.53576498 5.74E-06 5.84E-05 LINC01806 54.47482918 -1.63950987 0.249993018 -6.558222637 5.45E-11 1.12E-09 VSTM2L 514.9769983 -1.638584262 0.118936675 -13.7769469 3.51E-43 5.37E-41 MOK 1032.046494 1.638576 0.097478807 16.80956151 2.08E-63 7.91E-61 TSPAN1 17.45523461 -1.63480172 0.341665202 -4.784806028 1.71E-06 1.93E-05 FAXDC2 52.03161808 -1.633810033 0.265014672 -6.164979543 7.05E-10 1.28E-08 ACSS1 337.535724 -1.631097151 0.121000261 -13.48011266 2.05E-41 2.92E-39 DLGAP5 294.1759844 1.62665707 0.128273616 12.68115079 7.52E-37 8.87E-35 LARGE2 6.292900294 1.626427356 0.361554967 4.498423491 6.85E-06 6.89E-05 PCYT1B 6.543400199 1.625593357 0.361267245 4.499697607 6.81E-06 6.85E-05 TSKS 181.61521 -1.618956773 0.163648104 -9.892914939 4.47E-23 2.44E-21 KIF2C 39.69708567 -1.617184393 0.274852961 -5.883816515 4.01E-09 6.76E-08 ZNF85 28.53287794 -1.616443581 0.309051332 -5.230340117 1.69E-07 2.27E-06 ARHGEF39 10.29214436 -1.612269761 0.365503642 -4.411090828 1.03E-05 9.87E-05 FLI1 29.60322538 -1.611245336 0.304939148 -5.283825799 1.27E-07 1.73E-06 FOXE1 418.4286795 1.608332844 0.128352633 12.53057932 5.08E-36 5.73E-34 CHACI 335.9609796 -1.601673865 0.154026411 -10.39869627 2.51E-25 1.52E-23 CDCA8 111.7612165 -1.598629599 0.191861922 -8.332187954 7.94E-17 2.85E-15 SERTAD4 1466.570029 1.597898386 0.105877431 15.09196407 1.83E-51 4.16E-49 ADAMTS1 5.825359557 -1.596976341 0.359604577 -4.440923285 8.96E-06 8.71E-05 ARL17B 9.437142448 -1.596893811 0.366300399 -4.359519711 1.30E-05 0.000122378 ADAMTS12 14.40237091 -1.595021345 0.352765947 -4.521471982 6.14E-06 6.22E-05 NA 763.7452248 -1.593202742 0.095226295 -16.73070177 7.83E-63 2.85E-60 PCNA 878.1750915 1.591499677 0.090987246 17.49145901 1.66E-68 7.37E-66 LYPD3 17.61703887 -1.588056728 0.342409569 -4.637886523 3.52E-06 3.73E-05 SNORD17 607.3518203 -1.582092555 0.109739738 -14.41676992 4.06E-47 7.47E-45 TACC3 21.14248123 -1.581728648 0.335321214 -4.717055114 2.39E-06 2.63E-05 ZNF732 5.793912915 -1.580683281 0.3590842 -4.401985056 1.07E-05 0.000102468 HLA-DQB2 6.611233937 1.580663266 0.364231317 4.339723659 1.43E-05 0.000132496 PLCB1 225.7439332 -1.57905282 0.1844246 -8.562050931 1.11E-17 4.27E-16 HIST1H2AJ 105.6392088 -1.578599926 0.187485416 -8.419854527 3.77E-17 1.40E-15 KIF18A 466.1571922 -1.576418947 0.124249925 -12.68748408 6.94E-37 8.24E-35 FEN1 76.99680498 -1.573570167 0.221439732 -7.106087735 1.19E-12 2.99E-11 SKA1 235.2083453 1.572598127 0.144798989 10.86056011 1.78E-27 1.24E-25 KCNG1 26.58501856 1.572007526 0.319743761 4.916460356 8.81E-07 1.05E-05 NBPF3 26.98983965 1.571721031 0.306159668 5.133664542 2.84E-07 3.67E-06 IRF5 161.4557958 -1.571305988 0.160541659 -9.787528048 1.27E-22 6.86E-21 CCNF 96.639018 -1.568092062 0.198313811 -7.907124858 2.63E-15 8.35E-14 RFC2 16.69877159 1.566796659 0.344608388 4.546600464 5.45E-06 5.57E-05 HYAL1 10.29834603 1.566787084 0.364836545 4.294490515 1.75E-05 0.000159899 CAND2 99.15547596 -1.565380213 0.201172224 -7.781293975 7.18E-15 2.17E-13 ZNF724 240.3463396 1.565024208 0.146709993 10.66746838 1.45E-26 9.47E-25 LCE1C 263.7883169 -1.56468189 0.135491149 -11.54822212 7.54E-31 6.24E-29 BUB1 19.24204777 -1.56396976 0.335633671 -4.659752274 3.17E-06 3.40E-05 CSF1 116.6434989 -1.562941135 0.190672633 -8.196987216 2.46E-16 8.59E-15 POLA2 24.16471394 1.561896556 0.314636891 4.964124036 6.90E-07 8.37E-06 SAMD3 6.41124827 1.56151759 0.359395275 4.344847295 1.39E-05 0.00012977 NA 289.310227 -1.561136709 0.135087731 -11.55646553 6.85E-31 5.72E-29 CCNB2 76.82938117 -1.559271944 0.228388954 -6.827265128 8.65E-12 1.99E-10 MEF2C 7.86677926 1.558361323 0.366613264 4.250695422 2.13E-05 0.000191622 ROBO2 95.11859763 -1.558000922 0.210355468 -7.406514948 1.30E-13 3.55E-12 RAD51 9.530776103 -1.556478061 0.365705984 -4.256091317 2.08E-05 0.000187572 ZBTB7C 419.1547228 -1.551080765 0.124721636 -12.43634077 1.66E-35 1.79E-33 AURKB 222.9897496 1.55077099 0.154207118 10.05641643 8.61E-24 4.91E-22 CPA4 170.8839925 1.550750159 0.156646338 9.899689861 4.18E-23 2.30E-21 GJB6 63.63540568 -1.550571443 0.23314943 -6.650547862 2.92E-11 6.20E-10 CDC25C 48.3406719 1.550137675 0.273215502 5.673681259 1.40E-08 2.19E-07 CRIP2 8.551171635 1.54684843 0.366501236 4.220581758 2.44E-05 0.000215905 COX7A1 8.980914336 1.545958243 0.366602812 4.216984139 2.48E-05 0.000219022 TSLP 231.6366404 -1.543280537 0.14125429 -10.92554808 8.70E-28 6.23E-26 GPNMB 13.23083348 -1.54161959 0.355696521 -4.334086777 1.46E-05 0.000135476 NA 1581.637424 1.541601904 0.098607826 15.6336669 4.29E-55 1.15E-52 IL1B 1107.745316 1.541448057 0.088205125 17.47571985 2.19E-68 9.46E-66 MAP1B 529.3965158 -1.541117853 0.106622579 -14.45395394 2.37E-47 4.51E-45 SGO2 568.1159363 -1.540574751 0.10327609 -14.91705146 2.55E-50 5.50E-48 HMMR 85.3814292 -1.538921258 0.209898941 -7.331724731 2.27E-13 6.10E-12 DDIAS 66.74534185 -1.538022605 0.22691894 -6.777850287 1.22E-11 2.74E-10 CENPA 22.86123596 -1.53138216 0.320670082 -4.775569174 1.79E-06 2.01E-05 ARHGAP24 34.1631309 -1.527468466 0.288780515 -5.289375106 1.23E-07 1.69E-06 CXCL11 231.8736012 -1.525718245 0.145005063 -10.52182743 6.85E-26 4.33E-24 ZWINT 330.7528026 -1.525250076 0.128755728 -11.84607548 2.26E-32 2.01E-30 TYMS 898.5169208 1.524207251 0.231348795 6.588351803 4.45E-11 9.27E-10 PI3 65.42208502 -1.52329683 0.225958045 -6.74150295 1.57E-11 3.44E-10 POLE2 45.38560085 1.522942595 0.278795827 5.462573128 4.69E-08 6.87E-07 COL6A1 118.7968703 -1.521119133 0.187659138 -8.105755734 5.24E-16 1.78E-14 ACAA2 3526.061425 1.520087505 0.072389396 20.99875929 6.73E-98 7.88E-95 PYGB 89.41217093 1.512024776 0.207240531 7.295989675 2.96E-13 7.85E-12 SLIT2 914.3814823 1.511006657 0.103463103 14.60430447 2.64E-48 5.20E-46 SERPINB1 450.5008333 -1.510546632 0.116388792 -12.97845441 1.62E-38 2.06E-36 ARHGAP11A 214.4033311 -1.509580905 0.142064704 -10.62600955 2.26E-26 1.46E-24 KIF11 231.8596215 1.508298567 0.154501415 9.762360871 1.63E-22 8.69E-21 TNFRSF25 6.333988624 -1.506248769 0.361128231 -4.1709527 3.03E-05 0.000262794 HSD17B3 18.44594581 1.505706907 0.355624926 4.233974611 2.30E-05 0.000204651 LCE2C 42.02570528 -1.505015117 0.26804235 -5.614840771 1.97E-08 3.00E-07 C20orfl97 17.24900867 1.50311714 0.340828379 4.410187746 1.03E-05 9.90E-05 TH 18.48078592 1.502961172 0.335774563 4.476101937 7.60E-06 7.56E-05 ADSSL1 5.781342427 1.502218067 0.357993365 4.196217619 2.71E-05 0.000237527 NA 9.393725569 -1.500733479 0.366265808 -4.097388967 4.18E-05 0.000352493 CXCL10 326.7822287 1.499697964 0.134883853 11.11843954 1.02E-28 7.70E-27 XAF1 38.41236831 -1.499516483 0.277475951 -5.404131335 6.51E-08 9.33E-07 NA 45.88169463 1.499073129 0.261556493 5.731355053 9.96E-09 1.60E-07 ZNF704 275.6937066 -1.498624263 0.131093296 -11.43173836 2.90E-30 2.32E-28 BUB1B 11.68217886 -1.498516611 0.360646902 -4.155079673 3.25E-05 0.000279944 CYP1A1 333.5330032 -1.496239013 0.132105065 -11.32612903 9.74E-30 7.56E-28 NLRP2 134.2751816 1.496191906 0.174014121 8.598106281 8.10E-18 3.15E-16 MMP2 7.301162211 -1.49178312 0.365692319 -4.079339501 4.52E-05 0.000377125 MTCO3P12 1089.480616 1.489876422 0.100965391 14.7563082 2.80E-49 5.81E-47 SERPINB2 11.96818952 1.489431411 0.362834422 4.104989276 4.04E-05 0.000342159 FAM110B 374.1417045 -1.488823097 0.123559243 -12.0494676 1.95E-33 1.88E-31 CHAF1A 175.162267 -1.487825642 0.156980347 -9.477782853 2.60E-21 1.28E-19 TTK 252.8019722 -1.486727267 0.137090289 -10.8448766 2.11E-27 1.46E-25 KIF4A 107.1972813 1.485402017 0.213615862 6.953612931 3.56E-12 8.55E-11 KRT13 95.02252835 -1.484130342 0.195157553 -7.604780427 2.85E-14 8.26E-13 CENPN 188.7429338 -1.483834812 0.171615588 -8.6462706 5.32E-18 2.11E-16 TRIP13 7.89671238 -1.483417905 0.366384626 -4.048799544 5.15E-05 0.000424252 ETV7 19.77988829 1.483365616 0.331028335 4.481083513 7.43E-06 7.40E-05 FAAH 47.49108737 -1.480032307 0.258874924 -5.717171386 1.08E-08 1.72E-07 MNS1 48.67852418 -1.478793405 0.264111332 -5.599128941 2.15E-08 3.27E-07 DSCC1 25.58392481 1.478122468 0.31207214 4.736476861 2.17E-06 2.41E-05 LRRC6 88.74630306 -1.47622337 0.214532941 -6.881103508 5.94E-12 1.39E-10 POLQ 147.2519163 -1.476007563 0.168554286 -8.75686758 2.01E-18 8.14E-17 TCF19 6.409321726 -1.475530808 0.36377243 -4.056191966 4.99E-05 0.000412297 PYHIN1 5.59930165 1.474264235 0.357125399 4.128141655 3.66E-05 0.000312213 RIPPLY3 610.5057369 -1.474156826 0.098781934 -14.92334438 2.32E-50 5.08E-48 GMNN 75.09756961 -1.471328707 0.217881199 -6.75289431 1.45E-11 3.20E-10 PLK4 5.439530898 1.469036869 0.357134414 4.113400478 3.90E-05 0.000331119 PRMT8 138.1530225 -1.467605628 0.174281691 -8.420882443 3.74E-17 1.39E-15 NEK2 72.27500757 -1.467381313 0.226505359 -6.478351409 9.27E-11 1.86E-09 IL31RA 41.28697957 -1.467259466 0.273882637 -5.357256242 8.45E-08 1.19E-06 LMCD1 340.1349425 -1.462447907 0.129978342 -11.25147381 2.28E-29 1.76E-27 FANCD2 670.342867 -1.461408703 0.097595396 -14.97415624 1.08E-50 2.43E-48 CEP55 27.11600428 -1.460412332 0.302794843 -4.823108335 1.41E-06 1.62E-05 KLRG2 47.18706769 1.460155638 0.273068949 5.347204953 8.93E-08 1.25E-06 THNSL2 89.31333068 1.457570767 0.212819958 6.848844334 7.44E-12 1.72E-10 EPB41L4A 225.5473559 1.455496996 0.157900433 9.217815121 3.03E-20 1.40E-18 MDK 351.1623888 -1.455057996 0.126007795 -11.54736492 7.61E-31 6.27E-29 H2AFX 5.015941525 -1.454307612 0.355196694 -4.094372603 4.23E-05 0.000356111 DAW1 123.8364364 -1.451638675 0.179680618 -8.078994218 6.53E-16 2.19E-14 THEM6 626.9910581 -1.450011425 0.100257151 -14.46292273 2.08E-47 4.00E-45 NUSAP1 348.1892284 -1.449434495 0.119173887 -12.1623498 4.93E-34 4.99E-32 CDCA2 8.372665051 -1.449298318 0.366619789 -3.95313718 7.71E-05 0.000609881 TRAC 7.553340763 1.448468103 0.365972905 3.957856125 7.56E-05 0.000599406 PRKN 101.1762277 1.447462297 0.196853111 7.353006965 1.94E-13 5.25E-12 LIMD2 5.767851591 1.447360402 0.361422288 4.004624091 6.21E-05 0.000501799 SLC47A1 741.1408588 -1.446770325 0.099618121 -14.5231641 8.64E-48 1.69E-45 CCNB1 4606.281859 1.445786539 0.06743782 21.43880898 5.81E-102 7.93E-99 PTHLH 8.37667671 1.443979567 0.36633361 3.941706487 8.09E-05 0.000636623 PNMA6A 771.2376706 1.44317752 0.100571275 14.34979834 1.07E-46 1.90E-44 STC2 1770.648659 1.44289678 0.103123757 13.99189494 1.75E-44 2.95E-42 ADAM8 9.403571983 -1.438932632 0.365776298 -3.933914361 8.36E-05 0.0006548 CAMK4 116.0519579 1.432768859 0.211810925 6.764376559 1.34E-11 3.00E-10 SULT2B1 56.03844001 -1.432304845 0.250633084 -5.714747718 1.10E-08 1.74E-07 MT-TC 31.47077057 1.429746545 0.302806415 4.721652094 2.34E-06 2.58E-05 AMT 381.1758696 1.429659535 0.134534239 10.62673372 2.24E-26 1.46E-24 MIR503HG 844.3806322 -1.42946143 0.099535035 -14.3613898 9.04E-47 1.63E-44 CBR1 494.0058573 1.425413595 0.119287996 11.9493465 6.54E-33 6.13E-31 RFLNB 355.670225 -1.423352869 0.119332133 -11.92765797 8.49E-33 7.77E-31 MCM5 278.9527987 1.422915152 0.130320667 10.9185687 9.40E-28 6.69E-26 ADGRL1 43.79601493 1.421718876 0.263178913 5.40210027 6.59E-08 9.42E-07 BAMBI 6.301981137 1.421335918 0.364186744 3.90276676 9.51E-05 0.000736649 ARHGEF25 9.091511448 1.420581013 0.366294002 3.878253552 0.000105209 0.00080132 IFNE 63.62115808 1.417161454 0.229800705 6.166915172 6.96E-10 1.27E-08 DBNDD1 12.40209918 1.41581324 0.359638603 3.936766595 8.26E-05 0.00064862 SLCO2A1 127.0800844 -1.415174462 0.181058878 -7.816100896 5.45E-15 1.67E-13 FHOD3 53.1925153 -1.408883099 0.240637879 -5.854785238 4.78E-09 7.97E-08 SPESP1 126.9526283 -1.407705634 0.179260023 -7.852869868 4.07E-15 1.27E-13 CIT 121.4335406 -1.406461138 0.18249196 -7.706975901 1.29E-14 3.83E-13 BLM 886.4941826 -1.401308072 0.095213123 -14.71759388 4.97E-49 1.02E-46 PRC1 490.727929 -1.400858095 0.123309754 -11.36048085 6.58E-30 5.18E-28 PEG10 113.5334938 -1.400517023 0.188014791 -7.448972574 9.41E-14 2.61E-12 MND1 220.1598434 -1.400118908 0.145933228 -9.594243395 8.45E-22 4.29E-20 NCAPG2 203.6926962 1.399954518 0.166143793 8.426162 3.57E-17 1.33E-15 FKBP10 152.5899766 -1.396476732 0.172408432 -8.099816901 5.50E-16 1.86E-14 SKA3 41.73345692 -1.39619567 0.269947113 -5.172108176 2.31E-07 3.03E-06 APLN 47.1894848 1.39304744 0.262116097 5.314619965 1.07E-07 1.48E-06 ZFHX2 5.236010531 -1.392429067 0.358767803 -3.88114278 0.000103967 0.000793704 SNORA23 64.65142639 -1.389772501 0.231357645 -6.007030804 1.89E-09 3.29E-08 CENPP 30.10037663 1.389506974 0.300243927 4.627926993 3.69E-06 3.90E-05 ECM2 11.67182176 -1.388232587 0.358282094 -3.874691508 0.00010676 0.000812377 ZNF726 213.6029067 -1.38760455 0.154404336 -8.986823739 2.54E-19 1.07E-17 CENPK 8.282231694 -1.386636275 0.366574188 -3.78268934 0.000155143 0.001133679 GAD1 42.4594558 -1.385508977 0.274187842 -5.053137911 4.35E-07 5.44E-06 MMP17 639.702499 -1.383597116 0.10407884 -13.29374072 2.52E-40 3.41E-38 IFIT1 10.53920144 -1.382850104 0.365260936 -3.785923888 0.000153139 0.001120531 CD70 41.13261441 -1.382445162 0.273441675 -5.055722249 4.29E-07 5.38E-06 NA 346.2386994 -1.381265194 0.136799445 -10.09700876 5.70E-24 3.29E-22 MCM2 6.352708376 -1.378855326 0.364662617 -3.78118091 0.000156086 0.001139554 NA 34.69377337 1.377414802 0.28356589 4.857477047 1.19E-06 1.39E-05 CREB3L1 648.3954433 1.376631872 0.10073287 13.6661635 1.62E-42 2.39E-40 PCK2 17.95352843 1.376422653 0.347043604 3.966137502 7.30E-05 0.000582631 LCE2B 13.26007088 -1.375178693 0.357008097 -3.851953788 0.000117179 0.000881426 HRCT1 28.79990637 1.374747017 0.303879003 4.523994751 6.07E-06 6.16E-05 MEST 12.94786953 -1.373873069 0.360925775 -3.806525225 0.000140933 0.001039579 NA 13.43994975 1.37367671 0.356095924 3.857603013 0.000114504 0.000864084 EFCAB13 446.8418452 -1.373357871 0.117744123 -11.66391861 1.95E-31 1.66E-29 VCAN 82.93939185 -1.373203324 0.214335038 -6.406807488 1.49E-10 2.91E-09 APOBEC3G 25.97550777 1.37280693 0.309990814 4.428540673 9.49E-06 9.18E-05 TMEM132B 53.35795072 1.372357116 0.289970475 4.732747762 2.22E-06 2.45E-05 KPRP 92.72746768 -1.371967369 0.198426036 -6.914250745 4.70E-12 1.11E-10 IQGAP3 4.58182126 -1.370121141 0.351933659 -3.893123338 9.90E-05 0.000760456 UPK3BL1 89.17385734 -1.368522921 0.222553453 -6.149187548 7.79E-10 1.41E-08 XRCC2 4.848761727 1.368443239 0.353557134 3.870500989 0.000108612 0.00082341 ZNF208 105.0209506 -1.367555513 0.197654031 -6.918935614 4.55E-12 1.08E-10 GAMT 13.30255434 -1.366212206 0.353357332 -3.86637571 0.000110465 0.000835523 HSH2D 10.65919471 1.365337971 0.362202646 3.769541681 0.000163548 0.001188204 KIF21B 7.982701693 1.365300786 0.366652394 3.723692543 0.00019633 0.001403961 FIBIN 87.27825577 -1.364945829 0.208113997 -6.558645009 5.43E-11 1.12E-09 DDAH1 60.96127134 -1.363302515 0.231947258 -5.877640152 4.16E-09 6.99E-08 ZGRF1 96.4956854 -1.359664847 0.196203521 -6.929869748 4.21E-12 1.01E-10 MMS22L 51.62178422 -1.358611314 0.24844927 -5.468365091 4.54E-08 6.66E-07 FANCB 27.86646187 -1.357211989 0.303595034 -4.470468336 7.80E-06 7.74E-05 NA 180.52331 -1.356656527 0.157655567 -8.605192661 7.62E-18 2.98E-16 MASTL 28.60148125 -1.356298265 0.304465554 -4.454685422 8.40E-06 8.24E-05 RAD54L 270.7611834 1.355556443 0.143650867 9.436465442 3.86E-21 1.89E-19 CLDN4 8.007112146 -1.354973644 0.366601131 -3.696043273 0.000218986 0.001542408 CDKN2B-AS1 161.30197 -1.354162554 0.168097155 -8.055832661 7.89E-16 2.63E-14 CDKN3 170.5099024 -1.353894725 0.156225403 -8.666290517 4.46E-18 1.78E-16 PRIM1 16.07875322 1.351557239 0.344971171 3.917884602 8.93E-05 0.000696566 TNRC6C-AS1 215.1200834 -1.347050389 0.148958252 -9.043140401 1.52E-19 6.60E-18 TK1 8.424242319 -1.346451687 0.366052144 -3.678305697 0.000234788 0.001632657 MTCO3P22 35.00676121 1.345233436 0.288054669 4.670062938 3.01E-06 3.25E-05 KYNU 9.611075011 1.34419572 0.364450626 3.688279354 0.000225776 0.001578695 TRO 47.03617619 -1.34253612 0.253563243 -5.294679561 1.19E-07 1.64E-06 BTN3A2 130.3853575 1.342160798 0.186236681 7.206747823 5.73E-13 1.48E-11 GOLGA8A 9.300714848 1.342160413 0.366512149 3.661980688 0.000250273 0.001726408 MAPT 108.2540101 1.342086071 0.187990648 7.139110829 9.39E-13 2.38E-11 HSPA2 874.3850791 -1.341886597 0.088425499 -15.17533534 5.15E-52 1.21E-49 GPSM2 1310.716084 1.341621945 0.086376262 15.53229926 2.10E-54 5.29E-52 ASNS 94.93004281 -1.340369718 0.203491912 -6.586845172 4.49E-11 9.35E-10 SELENOM 350.8814774 -1.339695126 0.125745229 -10.65404337 1.67E-26 1.09E-24 KNL1 261.2208985 -1.338043011 0.134492342 -9.948841667 2.55E-23 1.41E-21 NUF2 7.315904614 1.337197067 0.366348075 3.650072594 0.000262166 0.001800867 EDA 45.29893007 1.336236928 0.263143165 5.077984558 3.81E-07 4.82E-06 TRPV3 213.0097117 -1.335871158 0.150801564 -8.858470167 8.11E-19 3.34E-17 CENPU 41.52328023 1.335863294 0.273496282 4.884392886 1.04E-06 1.22E-05 SERPINI1 84.58809498 -1.335848628 0.228085409 -5.856791248 4.72E-09 7.88E-08 OIP5 2450.868771 -1.335781682 0.073258995 -18.23368835 2.79E-74 1.58E-71 MDM2 40.72423106 1.335274598 0.272304911 4.90360087 9.41E-07 1.11E-05 AZIN2 10.10962507 -1.334719218 0.362652824 -3.680432441 0.000232839 0.001621853 UBE25 10073.25016 1.334652529 0.079494396 16.78926554 2.92E-63 1.09E-60 CSTA 265.9798785 -1.334462778 0.152276263 -8.763432683 1.89E-18 7.70E-17 HIST1H1D 313.0053761 -1.334339233 0.133742949 -9.976894054 1.92E-23 1.07E-21 CENPW 224.3116629 -1.334145321 0.14947221 -8.925708135 4.43E-19 1.84E-17 NEFM 13.60091323 -1.333693775 0.35208505 -3.787987522 0.000151872 0.001111764 LINC00911 14.50210611 1.333089573 0.35444224 3.761091153 0.000169174 0.001225818 RAET1L 81.07543899 -1.332331084 0.207942828 -6.407199025 1.48E-10 2.90E-09 GAS2L3 19.65834162 -1.331762779 0.329158128 -4.045966563 5.21E-05 0.000428769 GPR3 146.3589318 -1.330627736 0.166941078 -7.970643008 1.58E-15 5.15E-14 WDR76 169.0052119 1.329203629 0.159120048 8.35346421 6.63E-17 2.41E-15 SH2D2A 40.41865787 -1.328130984 0.272736468 -4.869649415 1.12E-06 1.31E-05 SFXN2 4.930081662 -1.328094921 0.35681196 -3.72211436 0.000197562 0.001411535 MTND3P10 557.8152566 -1.327320483 0.106651186 -12.44543577 1.48E-35 1.61E-33 NCAPD2 254.9088894 -1.326352834 0.161060177 -8.235138298 1.79E-16 6.31E-15 PSMB9 9.448461056 -1.326102308 0.364281503 -3.640322926 0.000272296 0.0018642 NA 34.67283174 1.323608038 0.292506054 4.525062027 6.04E-06 6.13E-05 SH3D21 1526.339126 1.323597459 0.086382315 15.32255129 5.41E-53 1.28E-50 PMEPA1 72.39654593 1.323476535 0.218634525 6.053373931 1.42E-09 2.49E-08 GSTM4 41.65868524 1.322138026 0.286060794 4.621877782 3.80E-06 4.00E-05 NOTCH3 127.8734403 1.321841562 0.200785728 6.583344232 4.60E-11 9.55E-10 AHSA2P 332.0592503 -1.32142553 0.120280149 -10.98623121 4.45E-28 3.23E-26 CDCA7 13.87354994 1.321166321 0.34976041 3.777346675 0.000158508 0.001155213 TSPAN33 11.91537354 1.320473803 0.356773038 3.701159177 0.000214617 0.001516854 COX6B2 8.248322737 -1.318981983 0.366305665 -3.600768723 0.000317278 0.00213381 APOL3 56.79800705 1.318606519 0.239100888 5.514854131 3.49E-08 5.18E-07 ABCA7 6.974413475 1.318604513 0.366318826 3.599608916 0.000318696 0.002141591 PAMR1 16.54672328 1.316835335 0.345619832 3.810068787 0.000138928 0.001027103 BEGAIN 39.83049294 -1.316451277 0.285760941 -4.606827203 4.09E-06 4.27E-05 FBXO32 1858.38226 -1.315362187 0.073849916 -17.81128878 5.78E-71 2.87E-68 SP110 12.02715194 1.314137069 0.359530001 3.655152739 0.000257029 0.001769288 TLL1 295.2164035 -1.313917731 0.125753675 -10.44834461 1.49E-25 9.22E-24 KIF14 1133.519777 -1.312901791 0.084413296 -15.55325832 1.51E-54 3.87E-52 TPX2 137.4384981 -1.310723547 0.198331774 -6.608742099 3.88E-11 8.15E-10 MT-TP 56.54688129 1.308465296 0.241391378 5.420513803 5.94E-08 8.54E-07 OPLAH 8.471890117 1.308464019 0.366650865 3.568692027 0.000358768 0.002372908 CILP2 4.561433087 1.308265855 0.350952285 3.727759899 0.000193189 0.001383918 NA 56.11349707 1.307528948 0.257128318 5.085122319 3.67E-07 4.65E-06 FUT3 459.1625598 1.303468793 0.122015022 10.68285504 1.22E-26 8.12E-25 TAGLN 5.678667797 1.303370295 0.36229126 3.597575869 0.000321197 0.002156626 PCSK4 18.04658791 1.30255893 0.334598687 3.892899114 9.91E-05 0.000760802 SYCP2L 318.8189538 1.302325766 0.124363993 10.47188769 1.16E-25 7.24E-24 RPS6KA2 11.7184985 1.302126764 0.363351256 3.583658358 0.000338815 0.002256427 RASL10B 6.514021391 -1.301656636 0.365352886 -3.562738067 0.000367007 0.002420561 RTP4 17.96676913 1.301182876 0.33265502 3.911508312 9.17E-05 0.000713181 ZNF322 48.27456923 -1.299889113 0.253850766 -5.120682249 3.04E-07 3.92E-06 FAM171B 768.3151969 -1.298822637 0.102444941 -12.67825068 7.81E-37 9.13E-35 COL8A1 8.192781018 1.2986897 0.366463501 3.543844604 0.000394338 0.002571828 SMOC2 593.66388 -1.29864349 0.1009593 -12.86303972 7.27E-38 8.82E-36 MCM3 8300.758341 1.297931641 0.07890055 16.45022289 8.36E-61 2.79E-58 FERMT1 5.996255051 1.29505432 0.360298764 3.594390125 0.000325152 0.002177829 NA 709.1605242 -1.294193763 0.092024633 -14.06355805 6.36E-45 1.09E-42 AURKA 170.7870677 1.293584872 0.163503417 7.911668707 2.54E-15 8.06E-14 MYL9 14.18532493 1.292979668 0.349860491 3.695700723 0.000219281 0.001543412 ARHGAP45 891.9500306 1.292757804 0.093437774 13.8354945 1.56E-43 2.45E-41 IFNK 15.32843997 1.289149053 0.350747176 3.675436731 0.000237443 0.001646287 RBP7 48.82008448 1.287408975 0.25056742 5.137974337 2.78E-07 3.60E-06 CCDC149 81.18593756 -1.287153036 0.207082981 -6.215638905 5.11E-10 9.43E-09 CENPO 77.84330321 -1.286581056 0.211604116 -6.080132466 1.20E-09 2.13E-08 USP18 227.2272738 -1.286386736 0.152605443 -8.429494467 3.47E-17 1.30E-15 CENPH 17.39658191 -1.286335418 0.336275251 -3.825245584 0.000130642 0.000971541 PLIN4 14.11454955 -1.284398185 0.34945551 -3.675426907 0.000237452 0.001646287 NA 6.384281158 1.282308307 0.36359106 3.52678723 0.000420635 0.002722741 DUSP9 4.396280788 1.281409466 0.349757383 3.663709552 0.000248589 0.001716236 CFAP46 22.00772859 -1.279915488 0.325101858 -3.936967614 8.25E-05 0.000648388 SLITRK5 4.176012181 -1.279813834 0.347636783 -3.681468407 0.000231895 0.00161665 U2AF1 32.48928402 1.279516394 0.298445592 4.287268526 1.81E-05 0.000165001 LEMD1 765.6833183 -1.278141518 0.111218715 -11.49214427 1.44E-30 1.17E-28 MCM4 692.3908368 -1.277546222 0.100167581 -12.75408875 2.96E-37 3.54E-35 IFIT3 6.547806354 -1.277410671 0.364769221 -3.50196946 0.000461833 0.002959016 RIBC2 74.90416104 -1.277200563 0.211857315 -6.028588451 1.65E-09 2.89E-08 FANCA 5.531935325 1.27680239 0.36171747 3.529833355 0.000415821 0.002697982 BGN 1232.72572 -1.27631125 0.101155077 -12.6173721 1.69E-36 1.95E-34 KLK7 7.103182321 1.273527241 0.36567281 3.482696024 0.000496392 0.003148426 NACA3P 302.0297975 1.273211585 0.130607557 9.748376056 1.87E-22 9.94E-21 RSRP1 13.41035012 1.271662407 0.351813097 3.614596549 0.000300816 0.002032274 DAPK2 32.18016403 -1.271157346 0.285712433 -4.449079562 8.62E-06 8.42E-05 NA 401.2878388 -1.270431908 0.116694052 -10.88686086 1.33E-27 9.40E-26 FBN2 64.49345913 1.269494545 0.234795118 5.406818312 6.42E-08 9.20E-07 PABPC1L 8.181153968 -1.267708504 0.366633556 -3.457699063 0.00054481 0.00341585 BCL2A1 358.4674268 -1.266977427 0.129166364 -9.808880521 1.03E-22 5.59E-21 RFC3 541.0687793 -1.266188151 0.105081823 -12.04954492 1.95E-33 1.88E-31 VRK1 29.53513905 -1.26504827 0.303568092 -4.167263638 3.08E-05 0.0002668 RAB17 62.99129717 -1.264883132 0.232555654 -5.439055593 5.36E-08 7.76E-07 BAG2 86.75083905 1.264535619 0.20239366 6.24790134 4.16E-10 7.77E-09 PTPRM 77.47835607 -1.263052793 0.225836387 -5.59277808 2.23E-08 3.39E-07 SLC25A19 22.93583517 -1.262730545 0.313217255 -4.031484613 5.54E-05 0.000453538 NA 11.18053625 -1.261470865 0.359654749 -3.507449486 0.000452424 0.002903294 FAM49A 8.64519229 1.260529785 0.366510802 3.439270487 0.000583284 0.00362242 NA 120.5250094 -1.259407594 0.192689921 -6.535928736 6.32E-11 1.29E-09 SIPA1L3 118.986988 1.257652533 0.186038287 6.760181227 1.38E-11 3.07E-10 MYEF2 13.48504835 1.256868679 0.352426972 3.566323745 0.000362024 0.002391549 RNF208 3650.461673 1.256617448 0.087250823 14.4023564 5.00E-47 9.10E-45 KRT16 8.530519353 -1.256492175 0.366186134 -3.431293703 0.00060071 0.003710946 DPYD 37.42764575 -1.256101575 0.286767004 -4.380216542 1.19E-05 0.000112083 EME1 80.94848389 -1.254874343 0.212619702 -5.901966422 3.59E-09 6.08E-08 PAX8 13.76568932 -1.254639715 0.359889184 -3.486183439 0.000489965 0.003117318 NA 13.45236837 1.253865637 0.35321573 3.549857866 0.000385439 0.002518807 TMEM121 35.31783269 -1.253572385 0.300362476 -4.173531933 3.00E-05 0.000260248 SLC16A7 813.6016737 -1.253289048 0.102331744 -12.24731448 1.74E-34 1.79E-32 ASPM 414.7921774 1.252877064 0.115876196 10.81220393 3.01E-27 2.08E-25 MTSS1 156.8439865 1.252066542 0.165807735 7.551315643 4.31E-14 1.24E-12 LENG8 15.45767903 -1.252048765 0.344497849 -3.63441678 0.00027861 0.001902655 ZNF488 12.6134251 -1.251999134 0.35961076 -3.481539689 0.00049854 0.003159482 RPTN 6.33704959 -1.251499777 0.364607171 -3.43246068 0.000598131 0.003697801 KIAA1324 147.9801395 -1.251404158 0.169920532 -7.364643616 1.78E-13 4.82E-12 RBL1 165.7917753 1.249483618 0.179470137 6.962069771 3.35E-12 8.07E-11 ARRDC3 84.8885445 -1.249348439 0.20567529 -6.07437304 1.24E-09 2.21E-08 PHEX 422.4860263 -1.247057724 0.117832931 -10.58326999 3.56E-26 2.30E-24 ANO4 53.8946185 1.246325814 0.248452591 5.016352661 5.27E-07 6.51E-06 GPRC5C 72.40453006 -1.246264941 0.220164503 -5.660607973 1.51E-08 2.35E-07 LETM2 42.72755021 1.245961808 0.266839149 4.669336608 3.02E-06 3.26E-05 SPOCK1 38.39605585 1.244396872 0.285883843 4.352805871 1.34E-05 0.000125755 S1PR1 266.7174704 -1.243762983 0.132848699 -9.362251906 7.81E-21 3.75E-19 GPX3 29.71312932 1.243580472 0.295428775 4.209408752 2.56E-05 0.0002254 NA 50.58241556 -1.241961643 0.267734945 -4.638773035 3.50E-06 3.72E-05 PIR 25.07577179 1.241867833 0.318659701 3.897159979 9.73E-05 0.000750359 NA 9.633488499 1.238723089 0.365125711 3.392593428 0.000692343 0.004196322 LCE2D 4.238041384 1.237293113 0.347429912 3.561274003 0.00036906 0.002431164 ALDH1A1 46.83579134 -1.236877692 0.257692191 -4.799826063 1.59E-06 1.80E-05 NKX3-1 70.78042 1.236678647 0.234763765 5.267757773 1.38E-07 1.88E-06 CEP112 75.75058485 1.236506332 0.226417767 5.461171832 4.73E-08 6.91E-07 PLXDC2 99.39007955 1.236460279 0.195709384 6.317838489 2.65E-10 5.07E-09 C4orf33 17.87652601 1.236402873 0.340284642 3.633437183 0.000279671 0.001906816 DMTN 22.54575737 1.235963392 0.324125484 3.813224981 0.000137165 0.001015444 SLC29A4 6.188539223 1.235220256 0.363347811 3.399553314 0.00067496 0.004106154 NA 11.0923246 1.233802634 0.361288456 3.415007074 0.000637804 0.003901843 PODXL2 30.6474591 -1.233618802 0.295133717 -4.179864006 2.92E-05 0.000253644 CNTNAP2 277.2453125 -1.232990496 0.127935507 -9.637594161 5.55E-22 2.87E-20 CKS2 145.89235 -1.232674963 0.163401518 -7.543840357 4.56E-14 1.31E-12 STIL 11.9751199 1.231748547 0.359680699 3.424561144 0.000615793 0.00378699 CDHR1 42.12421175 -1.230281612 0.288175163 -4.269214593 1.96E-05 0.000177456 FAM13C 46.93541392 -1.230106017 0.257100689 -4.784530232 1.71E-06 1.93E-05 TNS3 464.8328179 1.229961638 0.110082947 11.17304426 5.53E-29 4.23E-27 SYNPO 278.2517974 -1.229541056 0.134926076 -9.112701502 8.04E-20 3.54E-18 FANCI 25.06289793 -1.228219027 0.314200455 -3.909030075 9.27E-05 0.000719171 NA 219.7165483 -1.226317702 0.142799325 -8.587699583 8.87E-18 3.44E-16 ID3 19.29208535 1.22568211 0.329789765 3.716555946 0.000201957 0.001438548 CCDC7 2260.392877 -1.224394813 0.075848785 -16.14257644 1.28E-58 3.89E-56 CYGB 144.2947064 -1.224092915 0.169906177 -7.204522717 5.82E-13 1.50E-11 CDC25A 95.13107217 -1.22398983 0.203390061 -6.017943173 1.77E-09 3.08E-08 SYT8 6.962476366 1.223669146 0.366374845 3.339937672 0.000837972 0.004965097 GCK 33.6482199 -1.222546753 0.293698346 -4.162593254 3.15E-05 0.000271887 C17orf53 4.209329479 -1.22156721 0.343961034 -3.551469752 0.000383086 0.002509436 MINOS1P3 15.83231345 -1.220732754 0.34507675 -3.537568822 0.000403829 0.002625369 HS3ST3A1 502.6633443 1.219544808 0.11099421 10.98746329 4.39E-28 3.20E-26 MAP2 392.866991 -1.219328611 0.112995789 -10.79092078 3.80E-27 2.62E-25 FAM111A 16.72614712 1.218726935 0.350063376 3.481446552 0.000498713 0.003159482 LCE2A 842.5732879 1.218338035 0.091239219 13.3532274 1.13E-40 1.60E-38 LINC01133 1383.070213 -1.218036598 0.084480202 -14.41801246 3.99E-47 7.42E-45 KIF20B 13.74529717 1.216312571 0.354319159 3.432816267 0.000597347 0.003694349 LINC02057 38.02129609 -1.214873291 0.271974603 -4.466863008 7.94E-06 7.86E-05 DOCK10 7.509573447 -1.213179569 0.366611035 -3.309173633 0.000935718 0.00549064 GBGT1 31.74289489 -1.212279425 0.296515554 -4.088417659 4.34E-05 0.000364526 TRANK1 149.9473451 -1.210286654 0.180650459 -6.699604663 2.09E-11 4.52E-10 BRIP1 40.96766613 1.20941171 0.285533304 4.235623983 2.28E-05 0.000203265 PTP4A3 761.92835 1.207022002 0.103664814 11.64350713 2.48E-31 2.10E-29 PTPRZ1 4.092074585 1.206994895 0.345814392 3.490296886 0.000482484 0.0030745 HNMT 94.79206256 -1.206435257 0.207281055 -5.82028714 5.87E-09 9.71E-08 PSMC3IP 182.6405185 -1.205764823 0.160692865 -7.503536797 6.21E-14 1.75E-12 TTC12 599.3295523 -1.203252294 0.106219139 -11.32801774 9.53E-30 7.44E-28 CCBE1 8.751389078 -1.202585212 0.366065982 -3.2851597 0.001019246 0.005927663 HIST1H2AG 48.99125051 -1.201483353 0.246910973 -4.866058962 1.14E-06 1.33E-05 PLB1 32.83636234 1.20001158 0.296224337 4.05102293 5.10E-05 0.000421088 S100P 176.5737215 -1.199342171 0.152998694 -7.838904643 4.54E-15 1.41E-13 NCAPD3 8349.085459 1.196174911 0.177051753 6.756074933 1.42E-11 3.15E-10 GOS2 9170.215765 1.195849386 0.082877259 14.42916189 3.39E-47 6.39E-45 KRT6B 246.3320785 1.195051045 0.137500344 8.691258585 3.58E-18 1.43E-16 FAM171A1 184.7492774 1.194577068 0.155258568 7.694113669 1.42E-14 4.21E-13 H6PD 33.70339151 1.194330707 0.300691662 3.971944885 7.13E-05 0.000569715 LYPD5 252.0928238 -1.191598241 0.150016757 -7.943100941 1.97E-15 6.37E-14 PADI3 170.58114 1.191000554 0.168636721 7.062522003 1.64E-12 4.05E-11 SLC6A9 47.68639057 -1.190052651 0.267201448 -4.453765728 8.44E-06 8.26E-05 ZNF826P 141.4536326 -1.188572483 0.172609981 -6.885885007 5.74E-12 1.35E-10 TENT5A 3.906205441 1.187420144 0.344920485 3.442590959 0.00057617 0.003582313 TFAP2B 7.99486761 1.183710585 0.366633445 3.228594119 0.001244003 0.007037467 ALDH1A2 6.939748515 -1.18362791 0.366307958 -3.231237229 0.001232556 0.006977527 IRAK3 701.6260621 1.182190622 0.118175835 10.00365787 1.47E-23 8.21E-22 IVL 10.33627202 -1.181917877 0.363212621 -3.254066097 0.001137658 0.006521432 GLS2 147.8346926 -1.180706678 0.210389996 -5.611990594 2.00E-08 3.05E-07 HIST2H2AC 266.6215837 -1.180351316 0.142169212 -8.30243972 1.02E-16 3.63E-15 SLC43A3 27.27798079 1.179729764 0.30214401 3.904528055 9.44E-05 0.000731997 TNNI3 5369.266183 1.179269722 0.075802922 15.55704826 1.43E-54 3.71E-52 INHBA 559.138282 1.178124494 0.140797832 8.3674903 5.89E-17 2.15E-15 SLPI 56.78193633 1.178109296 0.261912024 4.498110763 6.86E-06 6.89E-05 LCE1B 165.0662592 1.177308757 0.160363819 7.341486175 2.11E-13 5.68E-12 FGFR3 16.86483869 -1.176298102 0.338403722 -3.476019985 0.000508914 0.003217887 CA2 955.7373791 1.176162965 0.091908617 12.79709133 1.70E-37 2.05E-35 HEPHL1 7.587313232 1.176018817 0.366449824 3.209221942 0.001330947 0.007447031 NA 830.009604 -1.174956714 0.088497642 -13.27670085 3.16E-40 4.24E-38 ANXA6 47.68186433 1.174500637 0.252435933 4.652668197 3.28E-06 3.50E-05 CLEC11A 130.4433134 -1.174093196 0.176572669 -6.649348425 2.94E-11 6.24E-10 RAB27B 4.593969435 1.172559151 0.352326601 3.328046043 0.000874574 0.005163296 LINC02562 5.648310862 1.17239661 0.361671737 3.241604169 0.00118859 0.006768393 SULT1E1 16.02231237 1.171645931 0.343447504 3.411426545 0.000646239 0.003946081 CFAP57 8.078792522 -1.170358616 0.366603525 -3.192436889 0.001410778 0.007826878 E2F2 30.39432908 1.170316943 0.311988593 3.751152986 0.000176023 0.001270951 IL23A 40.40427734 1.170187524 0.267821994 4.36927343 1.25E-05 0.000117307 TNRC6C 19.17945823 1.170099772 0.338993358 3.451689379 0.000557089 0.003475546 KRT1 8.790023041 -1.168594478 0.366326898 -3.190031866 0.001422571 0.007878966 NA 358.562897 -1.166720934 0.122391755 -9.532675887 1.53E-21 7.68E-20 MCM6 8.088404921 1.165729857 0.366058342 3.184546623 0.00144981 0.008000077 NA 370.5624879 -1.165266972 0.114753998 -10.15447825 3.16E-24 1.84E-22 BRCA1 70.44001502 1.163854841 0.220566642 5.276658486 1.32E-07 1.80E-06 THBS3 129.7307991 -1.163774336 0.175661919 -6.625080391 3.47E-11 7.31E-10 CMBL 57.30802769 1.16321445 0.257502387 4.517295803 6.26E-06 6.34E-05 NEIL1 4.89664037 -1.162967472 0.355407135 -3.272211948 0.001067095 0.00615809 NA 169.3696881 -1.162331044 0.15590077 -7.455582431 8.95E-14 2.49E-12 RFC5 25.2918421 -1.162095622 0.329347314 -3.528480648 0.000417953 0.00270859 WDR62 206.8003958 -1.162074497 0.148262306 -7.837963192 4.58E-15 1.42E-13 ORC6 5.543351414 -1.161402332 0.352753389 -3.292391702 0.000993391 0.005799976 RGCC 54.31855813 -1.160622398 0.239099953 -4.854130607 1.21E-06 1.41E-05 CD74 30.59709866 1.160137417 0.288843496 4.016491404 5.91E-05 0.000479327 AK5 3.645681333 -1.159784361 0.340815445 -3.402968905 0.000666579 0.004062708 HDGFP1 22.2185958 1.158882404 0.3202257 3.618955014 0.000295795 0.00200414 CTSK 36.81585878 -1.157472454 0.272968483 -4.240315368 2.23E-05 0.000199388 DNA2 248.1221721 1.156477122 0.136503534 8.472140518 2.41E-17 9.09E-16 TMEM200A 335.5523019 -1.156171465 0.118248632 -9.77746165 1.41E-22 7.56E-21 SAA1 6.737024736 -1.153998626 0.363875474 -3.171410853 0.001517004 0.008306511 MIR646HG 7.409607809 -1.152769518 0.36659515 -3.144530191 0.001663537 0.009012478 HIF3A 67.62487001 -1.151526606 0.222555863 -5.174101415 2.29E-07 3.01E-06 PSRC1 22.52487866 -1.150568795 0.322576258 -3.566811768 0.000361351 0.002388064 FOXL1 14.00213569 1.148476522 0.359094547 3.198256643 0.001382612 0.007694065 AMACR 201.7721606 1.147551141 0.153431722 7.479230018 7.48E-14 2.09E-12 FNDC4 33.96290994 1.147239888 0.294141462 3.900299808 9.61E-05 0.000742791 GAL 96.21461272 1.145792079 0.20727402 5.527909773 3.24E-08 4.83E-07 METRNL 14.848986 1.145499679 0.353858579 3.237168029 0.001207223 0.006855437 PAPPA 77.58689889 -1.145064106 0.218784453 -5.233754467 1.66E-07 2.23E-06 CENPI 49.36860103 -1.144887955 0.250085442 -4.577987209 4.69E-06 4.86E-05 SCN4B 8.898419687 1.144884215 0.365405772 3.133185908 0.001729199 0.009312776 CD86 23.55655921 1.143738205 0.321220269 3.560604098 0.000370003 0.002435417 BNC2 29.64382302 1.14365561 0.293150095 3.901262969 9.57E-05 0.00074054 GPR68 4.23376046 1.142928152 0.35089678 3.257163412 0.001125316 0.006457873 LINC00923 4.338589995 -1.142390127 0.34827249 -3.280161825 0.001037476 0.006012367 RPS26P3 441.7119798 1.141540902 0.117191156 9.740845122 2.02E-22 1.07E-20 TUBA1A 5695.769188 1.141453537 0.09525075 11.98366985 4.33E-33 4.10E-31 SPRR1B 22.23583768 1.141176346 0.326564188 3.494493238 0.000474963 0.003037203 GSDMB 19.18164618 1.140972379 0.328044249 3.478105107 0.000504972 0.003196659 WNT3 7.64481045 -1.140791124 0.366546875 -3.112265315 0.001856576 0.009888257 PAQR5 63.24835709 -1.140318856 0.230245312 -4.952625736 7.32E-07 8.84E-06 TEDC1 186.7627403 -1.14023235 0.151653584 -7.51866407 5.53E-14 1.58E-12 LNCAROD 578.1190238 -1.139833157 0.106476707 -10.70500012 9.64E-27 6.47E-25 HMGB2 4.057770347 -1.138115939 0.348896435 -3.262045198 0.001106115 0.006367352 SUSD5 56.01571744 1.137016667 0.252979343 4.49450399 6.97E-06 7.00E-05 CRYAB 302.170216 -1.136247267 0.154608323 -7.349198561 1.99E-13 5.39E-12 H19 15.1836841 -1.134809064 0.342075694 -3.317420926 0.000908526 0.005344483 TPK1 330.6442517 -1.133947465 0.126836275 -8.94024576 3.88E-19 1.63E-17 KIF23 29.56205561 1.133389226 0.296696483 3.82002919 0.000133436 0.000990521 PGGHG 28.7753477 -1.133363806 0.29244917 -3.875421523 0.00010644 0.000810321 TRAIP 2107.370978 1.132918487 0.099306631 11.40828644 3.80E-30 3.02E-28 UPP1 149.8260231 -1.132315181 0.168482047 -6.720687445 1.81E-11 3.94E-10 KIF15 3.784794772 1.132046525 0.341286734 3.316995396 0.000909911 0.005350709 CTNND2 207.594822 -1.131015533 0.145022154 -7.798915582 6.24E-15 1.90E-13 PHYHD1 305.2844192 1.130985308 0.123249427 9.176394037 4.46E-20 2.01E-18 IQCA1 53.51834962 1.130947664 0.254700236 4.44030866 8.98E-06 8.73E-05 RAET1E 232.6538256 1.128052613 0.138670985 8.134741464 4.13E-16 1.41E-14 RASSF6 44.1388473 1.127999441 0.263287764 4.284283571 1.83E-05 0.00016686 IGFL2 402.3981609 -1.12731928 0.117011517 -9.634259186 5.73E-22 2.94E-20 AFAP1 6.463945315 1.126771919 0.365721827 3.080953437 0.002063389 0.010813981 CYP2E1 1880.54812 -1.126529503 0.084395081 -13.34828401 1.21E-40 1.68E-38 RRM1 281.206243 -1.126424522 0.124953796 -9.014728294 1.97E-19 8.49E-18 GPR153 108.6515686 -1.126094082 0.182020292 -6.186640339 6.15E-10 1.13E-08 XRRA1 114.7364911 -1.125258669 0.191031565 -5.890433177 3.85E-09 6.50E-08 FANCG 16.26235857 1.124996245 0.343305651 3.276952305 0.001049341 0.006070807 RAET1G 588.8072745 1.124932561 0.106585902 10.55423407 4.86E-26 3.11E-24 GNAL 249.0262044 1.123759015 0.146739536 7.658188419 1.89E-14 5.52E-13 NFKBIZ 1323.220017 -1.123052111 0.083167218 -13.50354305 1.49E-41 2.14E-39 PRR11 3.990272107 -1.122665677 0.347881074 -3.227153652 0.001250283 0.007060132 RNU1-122P 372.1620506 -1.122615164 0.1220776 -9.19591444 3.72E-20 1.70E-18 BARD1 11.00619475 -1.122614309 0.362923378 -3.093254328 0.001979744 0.010428012 CEL 77.09615181 -1.12217499 0.214573453 -5.229794152 1.70E-07 2.28E-06 MRM3 358.4887477 1.121906708 0.122107264 9.187878516 4.01E-20 1.82E-18 SLC1A4 8.33281794 1.121324778 0.366437 3.060075204 0.002212814 0.011497791 APCDD1L-DT 13.78471621 1.120865973 0.348681958 3.214579786 0.001306356 0.00732194 SMPDL3B 12.95094029 -1.119970028 0.356598024 -3.140707327 0.001685404 0.009112861 TMPO-AS1 98.92197041 -1.119835383 0.198054504 -5.654177822 1.57E-08 2.43E-07 HAUS8 498.2780731 1.118907056 0.111458189 10.03880529 1.03E-23 5.79E-22 PTPRK 12.72086686 1.118640889 0.352724476 3.171429725 0.001516906 0.008306511 GRIN2D 29.98595721 -1.117522755 0.290279166 -3.849820743 0.000118204 0.000887507 CNIH3 8.584237897 1.117377123 0.365328609 3.058553572 0.002224083 0.011541702 GCNA 134.5969387 -1.11712152 0.170779949 -6.541292052 6.10E-11 1.25E-09 SLC37A2 10.50148372 -1.116774104 0.359924083 -3.102804613 0.001916962 0.010143922 KIAA1614- AS1 4.086015025 -1.116405209 0.347196742 -3.215482963 0.001302252 0.007303937 MAP2K6 4.283626928 1.11531508 0.349812613 3.18832151 0.001431013 0.007920302 GABRA3 33.12238019 -1.114323688 0.287220043 -3.879686376 0.000104591 0.000797727 ZNF90 18.5478728 -1.114029795 0.341585353 -3.26135118 0.001108826 0.006376237 IL15RA 62.19602554 -1.113725657 0.227028403 -4.905666621 9.31E-07 1.10E-05 DLEU1 203.3007417 1.110851972 0.143992031 7.714676766 1.21E-14 3.62E-13 WNT5A 691.4495322 -1.109849208 0.108157669 -10.26140092 1.05E-24 6.29E-23 NRP2 6261.205837 1.109305428 0.069356729 15.99420047 1.40E-57 4.03E-55 PHLDA1 14.73729954 -1.109272265 0.350431528 -3.16544653 0.001548451 0.008459362 CAMK2B 11.89009169 -1.109043318 0.359386673 -3.085933346 0.002029143 0.010651538 CRNN 18.09974898 1.108706302 0.332242391 3.337040463 0.000846756 0.005011708 NA 10.31620899 1.107792041 0.362377269 3.057013056 0.002235545 0.011590171 SEMA6A 23.10934272 -1.107492721 0.315348399 -3.511965574 0.000444806 0.00286336 CYSRT1 5.407785996 1.106756945 0.360198347 3.072631936 0.0021218 0.011088182 SULF1 4.401425792 -1.105546593 0.354288524 -3.120469666 0.001805629 0.009660881 NA 227.8707927 -1.105026225 0.13645535 -8.098079177 5.58E-16 1.88E-14 HERC5 4.293602515 1.103757683 0.34899724 3.162654473 0.001563378 0.008531918 GDA 6.474996047 1.102574778 0.364846726 3.022021846 0.002510924 0.012791192 NUDT16P1 596.5161224 -1.102453475 0.305913836 -3.603803901 0.000313594 0.002110766 RNU4ATAC 61.06336934 -1.100983162 0.234325271 -4.69852507 2.62E-06 2.85E-05 DHFR 7.925646452 1.100846509 0.366463007 3.003977176 0.002664753 0.01346181 RNFT2 23.19402302 1.099338226 0.314721861 3.493046919 0.000477543 0.003049002 ZFP3 17.93460815 1.098465078 0.332956344 3.299126439 0.000969862 0.005672707 C1orf210 9.750916719 1.098015271 0.364240605 3.01453286 0.002573753 0.013072729 NA 76.09017777 1.097992772 0.230428084 4.765012822 1.89E-06 2.11E-05 GABRE 2301.245347 1.097145117 0.079582011 13.78634577 3.08E-43 4.76E-41 HLA-A 18.02260945 1.096854724 0.336753382 3.257145382 0.001125388 0.006457873 SOCS2-AS1 36.78890695 1.096832854 0.278092318 3.944132148 8.01E-05 0.000630517 NA 190.0382203 -1.096621838 0.162333068 -6.755381707 1.42E-11 3.16E-10 UBE2T 64.70209546 -1.096225071 0.235980226 -4.645410713 3.39E-06 3.61E-05 TNS2 241.9474572 1.095984848 0.152569032 7.183534132 6.79E-13 1.74E-11 HOTAIRM1 39.94782505 1.095903094 0.270663483 4.048950677 5.14E-05 0.000424192 ULK4 18.63775483 1.09552548 0.335974682 3.260738195 0.001111226 0.006387794 NA 565.491649 1.094585909 0.107563046 10.1762264 2.53E-24 1.48E-22 SERPINB7 493.3318756 -1.094574559 0.112757976 -9.707291639 2.81E-22 1.47E-20 DIAPH3 45.47601634 -1.094437449 0.259543823 -4.216773249 2.48E-05 0.000219109 TROAP 772.3959168 1.093351058 0.110267301 9.915460379 3.57E-23 1.97E-21 WSB1 24.75538921 -1.092926386 0.306466951 -3.566212879 0.000362177 0.002391596 PRRT3-AS1 1110.090444 -1.092788 0.087394818 -12.50403656 7.10E-36 7.91E-34 PTTG1 32.87471884 -1.092009788 0.302164273 -3.613960631 0.000301555 0.002035589 ESPL1 327.9674622 -1.09155303 0.118547719 -9.20771007 3.33E-20 1.52E-18 CYB5A 4.85522516 1.091344356 0.355026633 3.073978837 0.002112244 0.011048817 LINC00894 64860.29683 1.090437203 0.098258776 11.09760622 1.29E-28 9.60E-27 MALAT1 36.44299862 1.090087693 0.282128386 3.863800129 0.000111637 0.000843608 LMF1 184.9783383 -1.088691619 0.149589051 -7.277883035 3.39E-13 8.93E-12 DEPDC1B 16.93827942 1.088478559 0.337923649 3.221078375 0.001277092 0.007180028 PBX4 144.9880208 -1.088215381 0.173943453 -6.256144534 3.95E-10 7.39E-09 KRBOX1 283.6181124 -1.087280322 0.161170819 -6.746136352 1.52E-11 3.35E-10 PDPR 3.833368369 -1.0869553 0.346009409 -3.141403881 0.0016814 0.009097217 NA 10.71787003 1.086323325 0.363950423 2.984811272 0.002837533 0.014190265 CHMP1B2P 91.40595107 1.085559229 0.196521894 5.523858992 3.32E-08 4.93E-07 CTSS 35.21857398 1.085239685 0.280111331 3.874315549 0.000106925 0.000813254 NBPF8 3.518959438 -1.085091492 0.335283797 -3.236337398 0.001210742 0.006865899 NA 1067.876374 -1.085047521 0.105425732 -10.29205584 7.65E-25 4.59E-23 CENPE 84.86369233 -1.084440776 0.208625596 -5.19802362 2.01E-07 2.67E-06 PAX8-AS1 22.99204449 -1.083231623 0.321593139 -3.368329392 0.000756252 0.00452839 RAD54B 376.1187743 -1.083220571 0.116839773 -9.27099178 1.84E-20 8.66E-19 ITGB3BP 60.35831333 -1.081999696 0.231764733 -4.668526065 3.03E-06 3.27E-05 MTFR2 15.05964672 1.081931748 0.346748266 3.120222523 0.001807145 0.009665834 PYROXD2 24.36655648 -1.081590834 0.312557991 -3.460448515 0.000539276 0.003387127 RPS26P13 47.26340852 1.081375665 0.257399027 4.201164537 2.66E-05 0.000233017 MT1F 567.2985222 -1.080888151 0.106413058 -10.15747668 3.07E-24 1.79E-22 CPT1A 29.42120214 -1.079039685 0.31166432 -3.462185489 0.000535808 0.00336849 HSD17B14 5.781107214 1.078964256 0.361452543 2.985078612 0.002835055 0.01418653 PCSK5 221.1500958 -1.078161899 0.144073756 -7.48340247 7.24E-14 2.04E-12 ANPEP 151.5716396 -1.076865997 0.170375716 -6.32053687 2.61E-10 4.99E-09 RMI2 99.18194249 1.076403821 0.194387901 5.537401326 3.07E-08 4.58E-07 LZTS3 316.3512976 1.075953251 0.128519617 8.371899013 5.67E-17 2.07E-15 ROBO3 168.3438078 -1.074754814 0.156346968 -6.874164729 6.24E-12 1.46E-10 GEN1 4.514511158 1.074721257 0.353924929 3.03657971 0.002392788 0.012292584 HSPB8 285.0059226 -1.074388067 0.134866073 -7.966333163 1.63E-15 5.32E-14 NUDT15 73.9976225 1.07413776 0.248369146 4.324763268 1.53E-05 0.000140698 RHCG 83.9133296 1.073973978 0.204428927 5.253532325 1.49E-07 2.02E-06 ITM2A 3.835239026 -1.073607886 0.345010806 -3.111809451 0.001859445 0.009898176 NA 585.9920271 -1.073195785 0.108645104 -9.877994924 5.19E-23 2.82E-21 PPT1 17.54709142 -1.073174797 0.331433517 -3.237979084 0.001203796 0.006843094 NA 8.705224417 -1.072441046 0.36485169 -2.939389004 0.0032886 0.015992027 HECW2-AS1 1894.469937 1.07240368 0.077400693 13.85522063 1.18E-43 1.88E-41 SMURF2 46.91797534 -1.072029304 0.25289604 -4.239011826 2.25E-05 0.00020044 SCLY 3055.306265 1.071272285 0.093083756 11.5086921 1.19E-30 9.67E-29 NEAT1 28.3291749 -1.07019971 0.307955103 -3.475180958 0.000510509 0.003226723 LAMP3 355.732424 -1.070139345 0.120279985 -8.897069178 5.73E-19 2.37E-17 WDHD1 75.46092136 1.06923253 0.214312818 4.989120771 6.07E-07 7.43E-06 VLDLR 13.41777491 1.067496246 0.352481359 3.028518298 0.002457562 0.012578019 TMEM81 374.7844838 -1.066177071 0.119327731 -8.934864187 4.08E-19 1.70E-17 CCDC18 246.6523689 -1.066168496 0.133707201 -7.973904841 1.54E-15 5.03E-14 TTC39C 6.537889331 1.064845253 0.36420467 2.923755073 0.003458367 0.016688788 PCSK1N 421.0723271 1.064739301 0.116508635 9.138715788 6.32E-20 2.81E-18 ZNF185 64.03004154 1.064478813 0.234074049 4.547615665 5.43E-06 5.56E-05 SMARCD3 6.018388733 1.062884246 0.364975025 2.912210898 0.003588802 0.017236983 NA 22.93018493 1.062635566 0.323442409 3.285393433 0.001018401 0.005926983 CHST10 351.5732139 1.062599427 0.132446872 8.022835188 1.03E-15 3.41E-14 LARP6 18.29141438 -1.061565357 0.328008918 -3.236391755 0.001210511 0.006865899 SNCG 5.560164726 1.060357559 0.357750937 2.963954663 0.00303713 0.01498714 MDGA1 1163.303906 -1.060350097 0.081895803 -12.94755115 2.43E-38 3.06E-36 NASP 209.8527768 -1.060246679 0.142541712 -7.438150327 1.02E-13 2.81E-12 CAVIN2 635.5788645 1.05867191 0.101553596 10.42476048 1.91E-25 1.16E-23 MRC2 73.6210676 -1.058400309 0.216753473 -4.882968168 1.05E-06 1.23E-05 SPAG5 9.478123313 1.058330452 0.363717878 2.909756477 0.003617105 0.01735257 WNT10A 3.27178656 -1.058302278 0.333699203 -3.171425849 0.001516926 0.008306511 SENCR 254.3088834 -1.058269523 0.133555082 -7.92384316 2.30E-15 7.35E-14 TMEM164 94.52087913 1.057899776 0.202349675 5.228077456 1.71E-07 2.29E-06 PLEKHO1 506.572828 1.057597898 0.110026772 9.612186909 7.10E-22 3.62E-20 ABCA1 28.44617041 1.057267185 0.302687123 3.492937445 0.000477738 0.003049002 RHBDL1 19.11323463 1.056072674 0.33478196 3.15450891 0.001607685 0.008744588 CCDC88B 16.36180329 -1.055871066 0.347060531 -3.042325389 0.00234758 0.012086863 NA 7.112164841 1.05548348 0.366516851 2.879767949 0.00397968 0.01878395 AKR1E2 230.2172425 -1.055471724 0.142956602 -7.383161798 1.55E-13 4.21E-12 ENOSF1 44.06296829 -1.0525134 0.257067417 -4.094308853 4.23E-05 0.000356111 TXNDC16 136.7221255 -1.051944331 0.172076648 -6.113231182 9.76E-10 1.75E-08 CCDC88C 202.1680517 -1.05114205 0.141414166 -7.43307463 1.06E-13 2.91E-12 RFC4 763.21636 1.05082024 0.095333453 11.02257615 2.97E-28 2.18E-26 CCDC80 18.40708084 1.048985833 0.329861701 3.180077681 0.001472356 0.008113557 LHX5 4.804942212 -1.048756177 0.356503565 -2.94178314 0.003263284 0.015897228 LINC01117 25.78523112 1.048169012 0.31314523 3.347229687 0.000816235 0.004853861 TPPP 7.888519178 -1.047557985 0.3666511 -2.857097617 0.004275343 0.019960943 ZNF695 155.4388274 -1.046834289 0.157074179 -6.664585444 2.65E-11 5.69E-10 NID1 58.5538766 1.046262317 0.234905976 4.453962116 8.43E-06 8.26E-05 CCDC18-A51 288.7148003 1.046124135 0.128393874 8.147772962 3.71E-16 1.28E-14 SETBP1 16.97689572 -1.045771821 0.338367828 -3.09063609 0.001997283 0.010504488 UBA7 71.93609383 1.045619733 0.219855463 4.755941552 1.98E-06 2.20E-05 RNF180 30.29474657 1.045287766 0.289067776 3.616064652 0.000299116 0.002024127 IL11 223.4247735 1.044921549 0.149494447 6.989701415 2.75E-12 6.73E-11 PYROXD1 14.8751097 1.044148702 0.345122137 3.025446911 0.00248266 0.012670846 LINC00649 815.2069991 1.043992173 0.097054777 10.75673147 5.51E-27 3.73E-25 MYADM 125.7868768 1.043480192 0.176488641 5.912449568 3.37E-09 5.73E-08 ECM1 141.8745152 1.043365397 0.165909873 6.288748096 3.20E-10 6.06E-09 DUOXA1 12.07693944 -1.043101548 0.355881648 -2.931034948 0.003378348 0.016370148 NA 864.0941425 -1.042053646 0.088797208 -11.73520734 8.41E-32 7.25E-30 TMPC 67047.45932 1.041113524 0.060262482 17.27631328 7.09E-67 2.83E-64 LAMC2 221.7545565 1.040901417 0.142001747 7.330201475 2.30E-13 6.16E-12 TMC6 138.4137836 1.040575586 0.170009264 6.120699322 9.32E-10 1.67E-08 RHBDL2 120.9204365 1.040481441 0.18289607 5.688921802 1.28E-08 2.01E-07 ARHGAP31 119.8607159 -1.039540684 0.180193121 -5.76903644 7.97E-09 1.30E-07 NRM 266.875406 -1.039292734 0.138246633 -7.517671237 5.58E-14 1.59E-12 HS3ST2 86.28554882 1.039199171 0.19959001 5.206669255 1.92E-07 2.55E-06 FANK1 259.7015144 1.039191371 0.138383866 7.509483605 5.94E-14 1.68E-12 PRDM1 38.32954385 -1.039013638 0.27696091 -3.751481164 0.000175793 0.001269848 SNORA12 1538.877129 -1.038587437 0.079911396 -12.99673748 1.28E-38 1.63E-36 SMC2 317.0337692 -1.038554914 0.131053484 -7.924664651 2.29E-15 7.32E-14 CENPQ 13.68805935 -1.037186751 0.346342653 -2.994683856 0.002747294 0.013831875 NA 618.9212743 1.036991604 0.101672911 10.19929103 2.00E-24 1.18E-22 PYCR1 10.34813076 1.036613757 0.364721518 2.842206189 0.004480251 0.020735239 RASGRP2 18.76761109 -1.035259818 0.330052113 -3.136655627 0.001708867 0.009225804 DNM3 189.302859 -1.034840623 0.147917779 -6.996053032 2.63E-12 6.45E-11 SLFN12 191.0871748 1.034745452 0.147460495 7.017102791 2.27E-12 5.58E-11 ID1 54.79580366 1.034087757 0.265555619 3.89405338 9.86E-05 0.000758612 NA 3.40154955 1.033750197 0.334282958 3.092440618 0.001985179 0.010450898 ESPNL 18.20229314 -1.033007357 0.33058579 -3.124778461 0.00177939 0.009554669 KIF24 28.8495746 1.032406661 0.297125213 3.474651821 0.000511517 0.003231847 NA 72.78088718 1.032219106 0.21901108 4.713090797 2.44E-06 2.67E-05 ASIC1 51.06548979 -1.032152712 0.243311354 -4.242106639 2.21E-05 0.000198019 SUV39H1 112.6137851 -1.032134799 0.185426544 -5.566273175 2.60E-08 3.92E-07 DYSF 179.9930537 -1.032032176 0.153467201 -6.724773575 1.76E-11 3.84E-10 SESN1 131.8496759 1.030213594 0.194589891 5.294281149 1.19E-07 1.64E-06 PLPPR2 886.1631323 1.029952928 0.101872688 10.11019685 4.98E-24 2.88E-22 C1orf74 42.01840445 1.02988227 0.266057916 3.870895044 0.000108436 0.000822461 LCE1E 197.9263827 -1.029457827 0.142896071 -7.20424166 5.84E-13 1.50E-11 UBR7 193.6978861 -1.02935952 0.150883203 -6.822227391 8.96E-12 2.06E-10 RAB7B 759.1167252 1.028631834 0.096313762 10.68000893 1.26E-26 8.34E-25 MAOA 54.07709016 -1.027862066 0.243337285 -4.22402208 2.40E-05 0.000212749 CHTF18 5.620912162 -1.027754629 0.36309948 -2.830504272 0.004647469 0.021375487 HIST1H2BM 167.0236604 1.027582621 0.162756887 6.313604557 2.73E-10 5.19E-09 GOLGA8B 535.4755993 -1.027306656 0.107832787 -9.526848768 1.62E-21 8.10E-20 GSR 188.8134248 1.026999888 0.174870883 5.872903865 4.28E-09 7.17E-08 PPP2R5B 58.85541071 1.025197662 0.239532908 4.279986702 1.87E-05 0.000169831 LURAP1L 5.833281619 1.024892386 0.359067498 2.854316783 0.00431295 0.020125053 CACNA2D1 3.291645814 1.024511251 0.333827932 3.068980014 0.00214791 0.011203185 PPBP 319.5109983 -1.024464315 0.12410429 -8.254866253 1.52E-16 5.37E-15 LINC00665 57.79244343 -1.024280191 0.237351286 -4.315460887 1.59E-05 0.000146345 CARD16 17.27689214 -1.023718517 0.339996675 -3.010966258 0.002604178 0.013184254 FRGIJP 111.0162775 1.023190421 0.205257951 4.984900302 6.20E-07 7.57E-06 KIFC2 87.81998949 1.022917349 0.216694443 4.720551836 2.35E-06 2.59E-05 GABBR1 14.37582888 -1.022898524 0.34811446 -2.938397116 0.003299141 0.016033768 C5 8.478188004 1.022492803 0.36501438 2.801239784 0.005090668 0.023102607 DSC1 27.37416794 -1.022216435 0.295019986 -3.464905713 0.000530417 0.003339673 NA 183.7928292 1.021421888 0.311332351 3.280808709 0.001035099 0.006002843 LCE3D 4.722175391 1.020963741 0.358067217 2.851318669 0.004353831 0.020275388 IL37 3.771760136 1.019892922 0.345022361 2.956019774 0.003116371 0.015295239 NA 184.2690947 1.019699727 0.171896099 5.932070205 2.99E-09 5.11E-08 MELTF 207.2226852 1.019395056 0.152473752 6.685708488 2.30E-11 4.96E-10 SERPINB13 1800.522954 1.018338849 0.082294942 12.37425801 3.60E-35 3.76E-33 TMEM154 63.2520462 -1.018220269 0.249279509 -4.084652895 4.41E-05 0.00036954 CMPK2 129.6616073 -1.018191587 0.175367163 -5.806056116 6.40E-09 1.05E-07 ARL4A 6.565541172 -1.017897617 0.365965813 -2.781400829 0.005412486 0.024194479 DAPL1 58.19726979 -1.0168268 0.229697584 -4.426806681 9.56E-06 9.24E-05 HOXD-AS2 275.0909719 -1.01641649 0.137174183 -7.409677764 1.27E-13 3.47E-12 IRF1 9.390555217 -1.016250035 0.364150949 -2.790738395 0.005258796 0.023694953 NA 185.4523567 -1.0161691 0.159043749 -6.389242613 1.67E-10 3.24E-09 MX2 10.80083972 -1.015775718 0.359126664 -2.828460874 0.004677242 0.021488293 NA 220.9454753 -1.015763171 0.14298854 -7.103808282 1.21E-12 3.04E-11 SLC29A1 132.0428796 -1.015667608 0.172314212 -5.894276483 3.76E-09 6.36E-08 FMN1 30.57655431 1.015429984 0.290355622 3.49719415 0.00047018 0.003008965 FAM171A2 90.77864692 1.014697477 0.203051796 4.997234675 5.82E-07 7.15E-06 UPK1B 146.4542942 1.014006018 0.186138411 5.447591466 5.11E-08 7.44E-07 MAPK12 7.91503683 -1.013655026 0.366347749 -2.766920305 0.005658859 0.025050503 NA 63.64461087 -1.013264374 0.229029027 -4.424174466 9.68E-06 9.32E-05 NA 8.940500898 1.013260909 0.364123421 2.782740275 0.005390194 0.024140936 AURKC 54.62505397 1.013224576 0.245568238 4.126040834 3.69E-05 0.00031475 RUNDC3A- AS1 15.73114391 -1.013109084 0.342641305 -2.95676286 0.003108871 0.015263002 NA 3.287040857 1.012847841 0.33278722 3.043529858 0.002338202 0.012042367 TDRD1 423.4817774 1.012508949 0.112446546 9.004357947 2.17E-19 9.28E-18 CCDC92 17.1633943 1.012311698 0.34297576 2.951554643 0.003161786 0.015457935 MFAP3L 13.8748548 -1.011865063 0.350386984 -2.887850037 0.003878848 0.018387488 BTN3A3 5.037508523 -1.011369994 0.359315383 -2.814713873 0.004882068 0.022310438 LINC00518 1292.819365 1.011260532 0.116227751 8.700680574 3.30E-18 1.32E-16 CD24 40.57252323 1.010888962 0.276067114 3.661750752 0.000250498 0.001727231 CMTM8 160.1137437 1.010617959 0.157715076 6.407871596 1.48E-10 2.89E-09 FCGRT 13.60116183 -1.010551692 0.348740459 -2.897718535 0.003758878 0.017931534 NA 4.773325915 -1.010375671 0.354172609 -2.852777561 0.004333895 0.020188284 RTCA-AS1 2408.975392 -1.010133335 0.071750122 -14.07848953 5.15E-45 8.88E-43 TFPI2 29.63867532 -1.009874785 0.291776271 -3.461127191 0.000537919 0.003380407 SNHG26 6.723358935 -1.009597406 0.365263156 -2.764027495 0.005709273 0.025238806 NA 11.47395663 1.009416172 0.360878947 2.797104626 0.005156283 0.023348643 BICDL2 202.6503912 -1.009258846 0.148562672 -6.793488773 1.09E-11 2.48E-10 MRI1 95.57007432 -1.00898385 0.20123882 -5.013862872 5.33E-07 6.58E-06 MYBL1 1701.099621 1.007678056 0.07951801 12.67232493 8.42E-37 9.78E-35 DLG1 8.914881087 1.007196049 0.364312448 2.764649015 0.005698407 0.025204376 SPRR4 30.85397041 1.00719405 0.293273324 3.434318668 0.000594045 0.003677627 COL13A1 17.03572567 -1.007159301 0.348416503 -2.890676225 0.003844139 0.018259941 SNORA31 5.201524039 1.006430382 0.361706234 2.782452406 0.005394978 0.024149159 NA 15.82037731 -1.006295883 0.339042098 -2.968055851 0.002996899 0.01483329 E2F8 16784.30701 1.003990295 0.077849482 12.89655714 4.71E-38 5.80E-36 LAMA3 14.10210352 1.00298529 0.348860376 2.875033562 0.004039846 0.019007694 EGFL8 36.63618482 1.002584723 0.306705472 3.268884365 0.001079724 0.006224181 GGT7 7.294974936 1.002267122 0.36665248 2.733561551 0.006265341 0.027357432 SEC31B 93.58946732 -1.001489346 0.199517257 -5.019562512 5.18E-07 6.41E-06 C3 1034.791179 -1.001457215 0.083858468 -11.94223123 7.13E-33 6.60E-31 CA12 12.56259131 -1.001393593 0.357721183 -2.799369007 0.005120259 0.023217602 SCAT8 5.458123772 1.000742686 0.361460365 2.768609736 0.005629602 0.024950381 SEMA6C

3. Discussion

Our study has identified the minimal number of mutations at endogenous loci implicated in human SCC that is sufficient for malignant transformation. These findings are consistent with sequencing data from human tumors (7-12) and data from normal appearing squamous epithelium (17,18) suggesting that no single gene mutation is sufficient for SCC tumor initiation. We have been able to reproduce an invasive phenotype into human dermis by employing human primary keratinocytes and using a model that better recapitulates the complex 3D architecture of human tissue than simpler in vitro systems. This system allowed us to unveil phenotypic differences not detectable in our own 2D culture, highlighting the importance of developing and employing more realistic models in the study of cancer biology. Our results indicate that in order to initiate squamous neoplasia, early mutations in keratinocytes favor cell cycle progression while also impeding keratinocyte differentiation. A partial disruption of the keratinocyte differentiation program is normally seen in pre-malignant lesions, while more extensive disruption of differentiation is often detected in SCC tumors (23). Previous studies have reported conflicting data on the effect of CDKN2A and TP53 mutations on keratinocyte differentiation (24,25). Here we identify that simultaneous mutations in both genes are sufficient to disrupt keratinocyte differentiation using primary human keratinocytes. Other studies have reported significant associations between aneuploidy and gene mutation rates (26). TP53 and CDKN2A are among those genes with mutations that are selected through a series of genome rearrangements or duplication events in a significant manner; suggesting that these mutations are required for tumor maintenance. Our results support the hypothesis that these mutations are the main driving events in SCC initiation. Importantly, mutations in NOTCH1 are sufficient to induce an EMT-like transcriptional program in CDKN2A-TP53-deficient keratinocytes, which enables tumorigenic invasion of human dermis. These results coincide with previous reports supporting the importance of NOTCH1 as a tumor suppressor (27). In addition, recently published data suggests that multiple other mutations/genomic alterations favor the inactivation of NOTCH signaling in NOTCH receptor wild-type SCC cases (28). Supporting these findings, our results demonstrate that alteration of the NOTCH signaling pathway is crucial for the development of SCC in a subset of tumors (FIG. 27 ). Our results further suggest that initiation of an EMT-like process in keratinocytes accompanies invasive progression of SCC. These data are consistent with a recent single-cell RNA-seq analysis of HNSCC, which reported the activation of an EMT-like program at the leading edge of malignant lesions (29).

The epithelium serves as a robust physical barrier between the environment and the body. Collectively, prior studies have reported a higher mutational burden in HNSCC and other SCC types when compared to other malignancies (15) and the tendency for normal aged epithelia to accumulate mutations without resulting in SCC formation (17,18). These studies report that mutations in NOTCH1 are relatively frequent in aged epidermis, but mutations in TP53 and, specifically in CDKN2A, are rare and were not detected in healthy epithelium (FIG. 28 ). Altogether, this implies that a very specific combination of genetic alterations is necessary to trigger SCC initiation. It may be the case that stratified squamous epithelia, like the skin and oral mucosa, have evolved to be robust to the oncogenic effects of single mutations, as they bear a large burden of exposure to mutagenic stimuli through sun exposure and ingestion.

Co-occurring mutations in CDKN2A, TP53, and NOTCH1 are detected in 12% of HNSCC (TCGA Data). Interestingly, the loss of function of these three driver gene mutations is sufficient to transform keratinocytes into their malignant counterparts. Both our genome and RNA sequencing results argue against the necessity of other mutations (e.g. TERT mutation, PI3KCA activation) to achieve this state. It is certainly the case that there are other combinations of mutations sufficient to induce tumorigenesis. It is possible that a subset of driver events is more important in initiating tumorigenesis, whereas other events may confer a selective advantage only after transformation. Future work is required to better define the rules of mutational progression in SCC.

Finally, it is discovered that the introduction of mutations in NOTCH1 results in a transcriptomic shift, which leads to an EMT-like phenotype. These results suggest that the upregulation of certain transcription factors is essential for the completion of this process in this context.

4. Sequences

The sequences mentioned in the disclosure but not disclosed elsewhere can be found in the following tables.

pSpCas9 vector components pUC (Origin of replication): SEQ ID ttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctacc NO: 01 aactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaaga actctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggact caagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctac accgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcgg cagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgac ttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaa U6 promoter (Guide RNA promoter) SEQ ID gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaaga NO: 02 tattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttac cgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggac CMV promoter (Cas9-GFP promoter) SEQ ID cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaatagtaacgccaatagggacttt NO:03 ccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacg tcaatgacggtaaatggcccgcctggcattgtgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcg ctattaccatg Cas9 gene SEQ ID gacaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaag NO: 04 aaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagcc gaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaac gagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcacccc atcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccg acaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaacccc gacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggc gtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaa gaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaac tgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccg ccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgat caagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttc ttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctgg aaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcag catcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaa agatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaa gagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaa cttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaag tgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagacca accggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaaga tcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggaca ttctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacg acaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaa gcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctga cctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccg ccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtga tcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatca aagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcaga atgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctga aggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgt gaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagag aggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacaga tcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtg tccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtg ggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcg ccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctgg ccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgcc accgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcc tgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtg gcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcat ggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctg cctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggcc ctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagct gtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatc tggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgacc aatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgcca ccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggcgac GFP gene SEQ ID gtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtg NO: 05 tccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggccc accctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccat gcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcg acaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactaca acagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggca gcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcaccc agtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcat ggacgagctgtacaag Ampicillin promoter SEQ ID cgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattga NO: 06 aaaaggaagagt Ampicillin (Bacteria selection marker) SEQ ID atgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagt NO: 07 aaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaa cgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcata cactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgc cataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatggg ggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaa tggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagt tgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtggaagccgcggtatcatt gcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagac agatcgctgagataggtgcctcactgattaagcattggtaa Guide RNA scaffold SEQ ID gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc NO: 08 Full pSpCas9-2A-BB-GFP vector sequence SEQ ID gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaag NO: 09 atattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgctt accgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgggtcttcgagaagacctgttttagagcta gaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttgttttagagctagaaatagcaag ttaaaataaggctagtccgtttttagcgcgtgcgccaattctgcagacaaatggctctagaggtacccgttacataacttacggtaaatggcc cgcctggctgaccgcccaacgacccccgcccattgacgtcaatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttac ggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattg tgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttc tgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcgggggggg ggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatc agagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcggg agtcgctgcgacgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactccc acaggtgagcgggcgggacggcccttctcctccgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggg gtattaatgtttaattacctggagcacctgcctgaaatcactttttttcaggttggaccggtgccaccatggactataaggaccacgacggag actacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaaggtcggtatccacggagtccc agcagccgacaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcc cagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcga aacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatctt cagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcg gcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggac agcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacct gaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgcc agcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcga gaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatg ccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttct ggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctc tatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagag attttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccat cctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacg gcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccg ggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgacc agaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatg accaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgac caaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaa gaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtg gaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacga ggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgtt cgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgg gacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacag cctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcag ccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacat cgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagaggg catcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctg cagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctt tctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagag gtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccg agagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggca cagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagc tggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccg tcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagat gatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattac cctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggatt ttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtc tatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccac cgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcacc atcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaa gctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaact ggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaac agctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgct aatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccct gaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggac gccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggcgacaaaaggccggcggc cacgaaaaaggccggccaggcaaaaaagaaaaaggaattcggcagtggagagggcagaggaagtctgctaacatgcggtgacgtc gaggagaatcctggcccagtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaa cggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaa gctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcac gacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccg aggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcac aagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgcc acaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgac aaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgcc gccgggatcactctcggcatggacgagctgtacaaggaattctaactagagctcgctgatcagcctcgactgtgccttctagttgccagcc atctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcat tgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctg gggagcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaagg tcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttct ccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtg gtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccgg ctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgat ggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactg gaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaaca aaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccag ccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccggga gctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatg ataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgt atccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcc tttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttac atcgaactggatctcaacagcctttggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgcta tgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccag tcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttactt ctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccgga gctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaa ctacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctg gctggtttattgctgataaatctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatggtaagccctcccgta tcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcatt ggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttg ataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatccttt ttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttcc gaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccg cctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttac cggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacc tacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacagg agagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgt gatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacat gt

Selection markers (including fluorescent gene reporters, antibiotic resistance genes and surface proteins) GFP SEQ ID NO: 317 Tdtomato SEQ ID atggtgagcaagggcgaggaggtcatcaaagagttcatgcgcttcaaggtgcgcatggagggctccatgaacggccacgagttcga NO: 10 gatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggcggccccctgccc ttcgcctgggacatcctgtccccccagttcatgtacggctccaaggcgtacgtgaagcaccccgccgacatccccgattacaagaag ctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggtctggtgaccgtgacccaggactcctccctg caggacggcacgctgatctacaaggtgaagatgcgcggcaccaacttcccccccgacggccccgtaatgcagaagaagaccatgg gctgggaggcctccaccgagcgcctgtacccccgcgacggcgtgctgaagggcgagatccaccaggccctgaagctgaaggac ggcggccactacctggtggagttcaagaccatctacatggccaagaagcccgtgcaactgcccggctactactacgtggacaccaa gctggacatcacctcccacaacgaggactacaccatcgtggaacagtacgagcgctccgagggccgccaccacctgttcctgggg catggcaccggcagcaccggcagcggcagctccggcaccgcctcctccgaggacaacaacatggccgtcatcaaagagttcatgc gcttcaaggtgcgcatggagggctccatgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggc acccagaccgccaagctgaaggtgaccaagggcggccccctgcccttcgcctgggacatcctgtccccccagttcatgtacggctc caaggcgtacgtgaagcaccccgccgacatccccgattacaagaagctgtccttccccgagggcttcaagtgggagcgcgtgatga acttcgaggacggcggtctggtgaccgtgacccaggactcctccctgcaggacggcacgctgatctacaaggtgaagatgcgcgg caccaacttcccccccgacggccccgtaatgcagaagaagaccatgggctgggaggcctccaccgagcgcctgtacccccgcga cggcgtgctgaagggcgagatccaccaggccctgaagctgaaggacggcggccactacctggtggagttcaagaccatctacatg gccaagaagcccgtgcaactgcccggctactactacgtggacaccaagctggacatcacctcccacaacgaggactacaccatcgt ggaacagtacgagcgctccgagggccgccaccacctgttcctgtacggcatggacgagctgtacaagtaa EBFP2 SEQ ID MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTG NO: 11 KLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKD DGTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMA VKQKNGIKVNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSV LSKDPNEKRDHMVLLEFRTAAGITLGMDELYK mKate2 SEQ ID atggtgagcgagctgattaaggagaacatgcacatgaagctgtacatggagggcaccgtgaacaaccaccacttcaagtgcacatcc NO: 12 gagggcgaaggcaagccctacgagggcacccagaccatgagaatcaaggcggtcgagggcggccctctccccttcgccttcgac atcctggctaccagcttcatgtacggcagcaaaaccttcatcaaccacacccagggcatccccgacttctttaagcagtccttccctga gggcttcacatgggagagagtcaccacatacgaagacgggggcgtgctgaccgctacccaggacaccagcctccaggacggctg cctcatctacaacgtcaagatcagaggggtgaacttcccatccaacggccctgtgatgcagaagaaaacactcggctgggaggcctc caccgagaccctgtaccccgctgacggcggcctggaaggcagagccgacatggccctgaagctcgtgggcgggggccacctgat ctgcaacttgaagaccacatacagatccaagaaacccgctaagaacctcaagatgcccggcgtctactatgtggacagaagactgga aagaatcaaggaggccgacaaagagacctacgtcgagcagcacgaggtggctgtggccagatactgcgacctccctagcaaactg gggcac mKO SEQ ID atggtgagtgtgattaaaccagagatgaagatgaggtactacatggacggctccgtcaatgggcatgagttcacaattgaaggtgaag NO: 13 gcacaggcagaccttacgagggacatcaagagatgacactacgcgtcacaatggccaagggcgggccaatgcctttcgcgtttgac ttagtgtcacacgtgttctgttacggccacagaccttttactaaatatccagaagagataccagactatttcaaacaagcatttcctg aaggcctgtcatgggaaaggtcgttggagttcgaagatggtgggtccgcttcagtcagtgcgcatataagccttagaggaaacacctt ctaccacaaatccaaatttactggggttaactttcctgccgatggtcctatcatgcaaaaccaaagtgttgattgggagccatcaacc gagaaaattactgccagcgacggagttctgaagggtgatgttacgatgtacctaaaacttgaaggaggcggcaatcacaaatgccaat tcaagactacttacaaggcggcaaaaaagattcttaaaatgccaggaagccattacatcagccatcgcctcgtcaggaaaaccgaagg caacattactgagctggtagaagatgcagtagctcattcc Puromycin SEQ ID atgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgcc NO: 14 gactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcg tcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcg ggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctc ctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggc agcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctc cccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagc ccggtgcctga DeltaLNGFR + SBP (Streptavidin binding peptide) SEQ ID atggacgaaaagaccacaggatggcgaggaggacacgtggtcgagggactggcaggagagctggaacagctgcgggctagact NO: 15 ggaacaccatcctcagggacagcgagagccaggaagtggagcgatcgcgaaggaagcctgccccacagggctgtacactcattct ggagaatgctgtaaagcttgtaacctgggagagggagtggcacagccatgcggagccaatcagactgtgtgcgagccttgtctgga ctccgtcacattctctgatgtggtcagtgccacagaaccttgcaagccatgtactgagtgcgtgggcctgcagtctatgagtgctcct tgtgtggaggctgacgatgcagtctgccggtgtgcatacggatactatcaggacgagactaccggcagatgtgaagcttgcagggtgt gtgaggcaggctcagggctggtctttagctgccaggataaacagaacaccgtgtgcgaggaatgtcctgacgggacatatagcgatga ggccaatcacgtggacccctgcctgccttgtactgtgtgcgaggataccgaaaggcagctgcgcgaatgtaccagatgggcagacg ccgagtgcgaggaaatcccagggcgatggattactcggtccaccccccctgaaggatcagacagcaccgcaccatctacacagga gccagaagcaccaccagagcaggatctgatcgcctccaccgtggctggcgtggtcacaactgtcatggggagctcccagccagtg gtcacccggggcaccacagataacctgattcccgtgtattgctccatcctggcagccgtcgtcgtcggactggtggcatacatcgcctt caagcggtga

Wild type genomic sequences genes: TP53 SEQ ID ctcattctccaggcttcagacctgtctccctcattcaaaaaatatttattatcgagctcttacttgctacccagcactgatataggcactcag NO: 307 gaatacaacaatgaataagatagtagaaaaattctatatcctcataaggcttacgtttccatgtactgaaagcaatgaacaaataaatctta tcagagtgataagggttgtgaaggagattaaataagatggtgtgatataaagtatctgggagaaaacgttagggtgtgatattacggaa agccttcctaaaaaatgacattttaactgatgagaagaaaggatccagctgagagcaaacgcaaaagctttcttccttccacccttcatat ttgacacaatgcaggattcctccaaaatgatttccaccaattctgccctcacagctctggcttgcagaattttccaccccaaaatgttagta tctacggcaccaggtcggcgagaatcctgactctgcaccctcctccccaactccatttcctttgcttcctccggcaggcggattacttgc ccttacttgtcatggcgactgtccagctttgtgccaggagcctcgcaggggttgatgggattggggttttcccctcccatgtgctcaaga ctggcgctaaaagttttgagcttctcaaaagtctagagccaccgtccagggagcaggtagctgctgggctccggggacactttgcgtt cgggctgggagcgtgctttccacgacggtgacacgcttccctggattgggtaagctcctgactgaacttgatgagtcctctctgagtca cgggctctcggctccgtgtattttcagctcgggaaaatcgctggggctgggggtggggcagtggggacttagcgagtttgggggtga gtgggatggaagcttggctagagggatcatcataggagttgcattgttgggagacctgggtgtagatgatggggatgttaggaccatc cgaactcaaagttgaacgcctaggcagaggagtggagctttggggaaccttgagccggcctaaagcgtacttctttgcacatccaccc ggtgctgggcgtagggaatccctgaaataaaagatgcacaaagcattgaggtctgagacttttggatctcgaaacattgagaactcata gctgtatattttagagcccatggcatcctagtgaaaactggggctccattccgaaatgatcatttgggggtgatccggggagcccaagc tgctaaggtcccacaacttccggacctttgtccttcctggagcgatctttccaggcagcccccggctccgctagatggagaaaatccaa ttgaaggctgtcagtcgtggaagtgagaagtgctaaaccaggggtttgcccgccaggccgaggaggaccgtcgcaatctgagagg cccggcagccctgttattgtttggctccacatttacatttctgcctcttgcagcagcatttccggtttctttttgccggagcagctcactattc acccgatgagaggggaggagagagagagaaaatgtcctttaggccggttcctcttacttggcagagggaggctgctattctccgcct gcatttctttttctggattacttagttatggcctttgcaaaggcaggggtatttgttttgatgcaaacctcaatccctccccttctttgaatg gtgtgccccaccccgcgggtcgcctgcaacctaggcggacgctaccatggcgtgagacagggagggaaagaagtgtgcagaaggca agcccggaggtattttcaagaatgagtatatctcatcttcccggaggaaaaaaaaaaagaatgggtacgtctgagaatcaaattttgaaa gagtgcaatgatgggtcgtttgataatttgtcggaaaaacaatctacctgttatctagctttgggctaggccattccagttccagacgcag gctgaacgtcgtgaagcggaaggggcgggcccgcaggcgtccgtgtggtcctccgtgcagccctccggcccgagccggttcttcc tggtaggaggcggaactcgaattcatttctcccgctgccccatctcttagctcgcggttgtttcattccgcagtttcttcccatgcacctgc cgcgtaccggccactttgtgccgtacttacgtcatctttttcctaaatcgaggtggcatttacacacagcgccagtgcacacagcaagtg cacaggaagatgagttttggcccctaaccgctccgtgatgcctaccaagtcacagacccttttcatcgtcccagaaacgtttcatcacgt ctcttcccagtcgattcccgaccccacctttattttgatctccataaccattttgcctgttggagaacttcatatagaatggaatcaggctgg gcgctgtggctcacgcctgcactttgggaggccgaggcgggcggattacttgaggataggagttccagaccagcgtggccaacgtg gtgaatccccgtctctactaaaaaatacaaaaattagctgggcgtggtgggtgcctgtaatcccagctattcgggagggtgaggcagg agaatcgcttgaacccgggaggcagaggttgcagtgagccaagatcgtgccactacactccagcctgggcgacaagaacgaaact ccgtctcaaaaaaaaggggggaatcatacattatgtgctcatttttgtcgggcttctgtccttcaatgtactgtctgacattcgttcatgttgt atatatcagtattttgctccttttcatttagtatagtccatcgattgtatatccgtccttttgatggccttttgagttgtttcccatttgcgg ttatgaaataaagctgctataaacattcttgtacaattctttttgtgatcatatgttttcgtgtttcttggagaaatacttaggaggggaatt gcgagtttggaagtaaaaagtagctgtattttgaactttttcagaagctctgagttttccagagcggttgtaccattttacactccaactagc aaggtatgggagttattatggttgtgccacagccttccggacattaggtattgtcagtctttctaatgtggtatatccttgtggttgtaattta cagttctctattgactaaggatgttcagcattttttcatgtgcctattggccattcgtattttgtttgtaaagtagctcttcgagtcttttacc tgttattttggttttttgtttgtttttattgttcagttgtgggactgctttatacattctggatacaagtcctttatcagatccatgtgtcgt gaatgttttcttctgatctgttgcttgcctatttgtttgctttacagagtttacagtatcttaagaggagtggatttatcttttttatgttca gtatttgccttgtcctgtttaggacatctttttttttttttttaaccccagggtcatgaagatattatcttacattttcttttaggaccttta tggttgtaagttttacagtaaggtccttgagccattaattaattcttaaaattaattgtttatggtgtgaggtgtaggagtcagtctctggt atctttcctgtatggaaatccagttattctgtctccacttgttgaaataggcttcctttctctactgaatgcttttaattttaattattttac agttggagtatagggctaccattttagtgctattttctttttttctttgttaatttttgagacagggactcacactgttgcccaggctagagt acaatggcacaatcaaggcttactgcagcctcgaacccctgggctcaagcagtcctctagcagcctcacgagtagctgggattactccaccac acccagctaactattttatttttttgtattgacaggatctcactatgttgcccaggctggtctcaaactgctggcctcaagctttcatcccat ctcggcctcccaaagtgctgggattacaggtgtgagccaccatgcctgacctcttagtgctattttctatttatctcctctgttctctgctct ctttaaacgttggaggaagaaacagtacccatcttacacaaactcttcagaaaacagaggaacagactgggcgcggtggctcatacctgtaat ctcagcactttggtacgctgaggcaggggatcatttgaggtcgggagttcgagaccagcctggccaacacggcgaaaccccatctctactaa aaatacaaaaagtagctaggcgtggtgacacatacctgtaatgccagttactcaggaggctgaggcacaagaatcccttgaacctgggaagcg gaggttgcagtgagccgagattgcgccactgcactccagcctgggcaacagagtgagaccctgtctcagaaaaaaaaagaaagaaagaaaaaa tagaggaatatttcccaacttgttttcgaagccagcataatcctggtaccaaaaccaaacaaggacattataagaaaagaaaatatagacca atattcctgttagcatagacatgcaacagctaaccaattttagcaaaccaaacctggtaatatagaaaaaaggataaataggccagtcgcggt ggctcacgcctgtaatcccagcactttgggaggctgaggcaggcagatcacttgaggtcaggagtttgagaccagcctgaccaacatggtga aaccccgtttctaataaaaatacaaaaatcaggctgggcacggtggctcacgcctgtaatcccagcactttgggaggccgaggtgggcaga tcacgaggtcaggagttcaagaccagcctgaccaatgtggtgaaacgccatctctactaaaaatacaaaaatcagccggtgtggtgg cacctgcctgtaatcccagctactcaggaggctgaggcagaattgcttgaacccgggaggcagaggttgcagtgagccaagatcgt gccactgcactccagcctgggcgacagagcaagacttcatctcaaaaaaaaaaaaaaattagctgggcatggtggtgggcacctga aatcccagctactcgggagtctgaggcaggagaatcgcttgaacccaggaggcagaagttgcactgagctgggatcacaccattgc actccagcctgggcaacagagtgagactccatctcaaaaaaagaaaaagaaaaaggataaatacattctaaccaaataatgtttatctc atgattgtagctgattcaacattcaaaaattggcctggtgcagtagctcaggcctgtaatcccaacattttaggaggctgaggcaggaa gatctcttgagcccaggatttcaagaccagcctgggcaacatagtcagactggtctttactggggggaaaaaaatcagtctgtgtaattc accacattaacaaagggaaacataaaaaccctatgatcatttcaacagatgtagcaaaagcagttaatgatattcaacacatatgcatga ttacaaaccaaccaacctcctagcaaactagggaaaggaaacttaacctagtttgataacagggcgtccacagtcggagttccactag cagcatacataatggtagaaaactcagtgctgccgggcgcggtggctcacgcctgtaatgccagcactttgggaggcctaggcggg cggatcacgaggtcaggagatcgagactgtcctgactagcatgctgaaaccccgtctctactaaaaatacaaaaacaaaaaattagcc gggcatggtggcgggcgcctatagtcccagctactcgggaggctgaggcgagagaatggcgtgaacccgggaggcggagcttgc agagcctagatcgtgccactgcactccagcctgggtgacagagtgagacttcgtctcaaaaaaaaaaaaaaaaaaaaaagaaaaga aaactcaacgctttttcctctaagatcaggaactagaaaaggatttgactctcacaacgttgataccatactggaggttttaaccaggcaa gaaaaagaaataatgagggccgggtgcggtggctcaggcctgtaatcccagcactttgggaagccgagacgggtggatcacgagg tcaggagatcgagaccatcctggctaacacggtgaaaccctgtctctactaaatatacaaaaaattagccgggcgtagtggcgggcg cctgtagtcccagctactcgggaggctgaggcaggagaatggcgtgaactcagggggcggagcttgcagtgagctgagatcgagc cactgcactccagcctgggcgacagagcaagactgtgtctcaaaaaaaaaaaaagaaaaagaaataatgattagtggcccgatgtct cacgcctataatcccagcactttgggaggccgaggtgggcagatcacctgaggtctggagttggagaccagcctgacaaagatggt gaaacctcgtctctattaaaatattaaaaaaatagccaggcgttggccgggtacagtggctcatgcctgtaatcccagcactttgggag gccgaggtgggtggatcacctgaggtcaggagttcaacaccagcctggccaacatggtgaaaccccatctctactaaaaatacaaaa attagccgggcgtagtggcgggcgcctgtaatcccagctacttgggaggcttaggcaggagaatcgcttgaacctgggaggcggag gttgtagtgagccgagattgcaccattgcactccagcctgggtgacaaaagcaaaaactccgtctcaaaaaaaaaagaattagccag gggtagtggtgaacgcctgtagtcccagctactcaggaggcagaggcaggagaatcacttgaacccaggaggcagaggttgcagt gagccgagattgtcccattgcactccagcctaggcgacaagagcaaaattccatgtcaaaaaaaaaaaaaaaaaaggaaagaaaaa aaataacgattagaaaggaagaaataaaacacattcacagccagtatgattctatacatacatgtcctaatggggccaggcgtggtgg ctcatgcctgtaatcctagcacttttaggaggctgaggcaggtggcttccctgggaccagcctggccaacatggtgaaaccccaactc taataaaaatacaaaaaatcagccaggcgtggtgacgggcacctctaatcccagctactcaggaggctgaggcaggagaattgcttg gacctgggaggcagaggttgcagtgagccgagatcgcgctattgcactccagcctgggcaacaagagtgaaactccggcagggtg tggtggcttacgcctgtaatcccagcacttcgggaggctgaggcaggccgatcacctgaggtcaggagtttgagaccaacctaacat ggtgaaaccccgtctctactaaaaatacaagaattagctgggtgtagtggtgggcgcctgtaatcccagctacttgggaggctgagac agaagaattgcttgaacccaggaggtggaggttgcagtgagctgagatcatgccattgcacaccacgccgggcaacagagcgagat tccgtctcaaaaaaaaaaaaaaagagtgaaactctatctcaaaaaaaaaaaaaagtcctaatggaaaatccataaaaagctaccaaaa ctaataaataaatatagcagggttgcaggttacagggcaatatagttatccctctatctgtaggggcttggttctgggactcctcacacac caaacccacagatgtctaagtcccatatataagacggtatagtatttggatttaacctacacatatcctcccatatagtttaaattatctctag attacttacattacccccatacaatgaaaatgctaatgtacatgcaagtatgtatgtaagtacttgtactatattgtttagggaatcactggac atataggccttcaagactgataccagcagccactgttaagattctggtcaggcctgcccctgtttggggtctcagttgatctcattgccttc ccacccagccaagggcacctgcatttctcttggctccctggccatttggaaggcctagttcagcctggcacatttgtatcctggcccact gatgctggtacccctgggaaggtcctgctctgaaaaacacggagattttagttgctactgaagatttgagagataaagacagggagac ctgtctgtagacctgtgtccctccaagtgggattgagactttgggccccccatttcaggacagcacctcctggcctgttgactgaataga tccctgaaggaggtgtacttgcattaatggagtgggggtgggagcagtaccacagatccgcactaacaatcacacagttctctctaga ataataatatagaacaagtgaaatagaacaattgcagaaagagctaacctttgttgagctcttactgtgtgcccagcactttcctcaactct acatttcccataatacacagagtactaggtaggccaggcttggtggctcacgcctgtaatcccagcactttaggaggccaaggggggt ggatcacctgaggtcgggagttcaagaccagcctgaccaacatggtgaaaccccgtctctactagaagtacaaaattagccaggtgt ggtggcacatgcttgtagtcctagctactcagcaggctgaggcaggagaatcatttgaatccgggaggaggttgcagtaagcggaga tagtgccactgtactccagcctgggcaataagagctgagactccgtctcaaaataaaataaaataaaataaaataaaataaaataaaat aaaaaaagaaaagagcctgccattaaaggagctgtttggtaggggatgttttgtcagtgcaaacaacagaaaagtgggctgggcaca gtggttcatgcctgtaatcccagcactttgggaggccaaggcgggcggatcacctgaagttgggagttcaagaccagcctgaccaat atggagaaaccccgtctctactaaaaatacaaaattagccgggcgcagtggcgcatgcctgtaatcccagctactcgggaggctgag gcaggagaatcgcttgaacctgggaggcagaggttgcggtgagccgagatcgcaccattgcactccagcctggacgagagcaaaa ctctgtctcaaaaaaaaaaaaaaacagaaaagtgtaacaaacacttacagtaggcatgtttcttagcaaatctgatgacaaatttggcat aaagaaagagagcatccctgaaaaaaaaaaaaagaaaaagaaagagagcatcctgcctgggcaacatagtgaaaccctgcctcta caaaaaaactcaaaaattggccgggtgcagtggctcacacctgtaatcccagcactttgggagtcggaggcgggaggatcacctga ggtcaggagttcgaaaccagcctggccaacatggcaaaaccccatctctactaaaaatacaaaaaattaatcaggcgcattggtggg cgcctgtaatcccagctactcaggaagttgaggcaagaggatcgcttgaatctgggaggtggaggttacagtgagtcgagatcacac cactgcactctagcctgggtgacagggcgagactccgtctccaaaaaaaaaaagaaaaagaaaaagactaaaaaattagccaggca ggcctctgtggtcccagctacttgggaggctgaggcaggagaatcactgagcccaggagtccgaggctgtagtgagccatgattgc accactgtaccctagcttgggcaacaaagcaagaccctgcctcaaaagaaaaaagaaagaaagaaagaacatggcgggccaggc acagtggctcacacctgtaatcccagcgctttgagaggccgaggcaggtggatcacaaggtcaggagttccacaccagcctggcca acatggtgaaaccctgtctctactaaaaatacaaaaaatcagccaggcatggtggcaggggcctgtaatcccagctactcgggaggc tgaggcaggagaattgcttgaaaccagaaggcagaggttgcagtgagcctagactgcaccactgcactccagcctgggcgaaaag agccaaactccatctcaaaaaacaaacaaaaaaacaaaacaaaagaaaacatggcaaagcctttgaaagcttgtctgggagaaggt gcgatgatagttgcataacttcgtgcaagatgctggtccacacaggggctgccccttgctctttctcgctctcttaacctctcatataacag gcttgtgtgttattcacatttattgagcccaagcaggtgcaaggcattgtgatctaatactttggtcagcaagacaacaagatagatcact gccctgcccttaggaagtgtatatgctattagaggaaacagataaaataaacaaggaaaagtatcagacaatgtaagtgctatgagaat gcaaatgaggtgatgtgaattaaaataggatgacttaaagtctgcacgggaaggagcctacccccatgttcctggctagccaaggaac caccagttgattagcagagaagggcagccagtctagctagagcttttggggaagagggagtggttgttaagagatgagattaaagaa gccgagacgggccattcgtgaggggtttgtaatgcagggctgaggagtgtccgaagagaatgggcaggtgagcggtgagacagtt gttcttccagaagctttgcagtgaaaggaatcaaagaaatggagccgtgtatcaggtggggaagggtgggggccaagggggtgtcc ttccccatacagagattgcaggctgagaatgactatatccttgttaacaggaggtgggagcagggcacggtagctcacacctgtaatct tggcactttaggaggctgaggcgggccgatcacctgaagtaaggagttcgagaccagcctggccaacatgcaaagccctgtctctac taaaaatacaaaaattagctgggtgtggtggtactcgcctgtaatcccagctactcgggagactgaggcaggagaatggcttgaaccc ggaaggtagaggttgcagtgagctgagatcatgccactgtgctccagcctaggtgacagagagagactccatctcaaaaaaaaaaa aaaaatacaggaagggagttgggaatagggtgcacatttaggaagtcttggggatttagtggtgggaaggttggaagtccctctctga ttgtcttttcctcaaagaagtgcatggctggtgaggggtggggcaggagtgcttgggttgtggtgaaacattggaagagagaatgtga agcagccattcttttcctgctccacaggaagccgagctgtctcagacactggcatggtgttgggggagggggttccttctctgcaggcc caggtgacccagggttggaagtgtctcatgctggatccccacttttcctcttgcagcagccagactgccttccgggtcactgccatgga ggagccgcagtcagatcctagcgtcgagccccctctgagtcaggaaacattttcagacctatggaaactgtgagtggatccattggaa gggcaggcccaccacccccaccccaaccccagccccctagcagagacctgtgggaagcgaaaattccatgggactgactttctgct cttgtctttcagacttcctgaaaacaacgttctggtaaggacaagggttgggctggggacctggagggctggggacctggagggctg gggggctggggggctgaggacctggtcctctgactgctcttttcacccatctacagtcccccttgccgtcccaagcaatggatgatttg atgctgtccccggacgatattgaacaatggttcactgaagacccaggtccagatgaagctcccagaatgccagaggctgctcccccc gtggcccctgcaccagcagctcctacaccggcggcccctgcaccagccccctcctggcccctgtcatcttctgtcccttcccagaaaa cctaccagggcagctacggtttccgtctgggcttcttgcattctgggacagccaagtctgtgacttgcacggtcagttgccctgagggg ctggcttccatgagacttcaatgcctggccgtatccccctgcatttcttttgtttggaactttgggattcctcttcaccctttggcttcctgt cagtgtttttttatagtttacccacttaatgtgtgatctctgactcctgtcccaaagttgaatattccccccttgaatttgggcttttatccat cccatcacaccctcagcatctctcctggggatgcagaacttttctttttcttcatccacgtgtattccttggcttttgaaaataagctcctga ccaggcttggtggctcacacctgcaatcccagcactctcaaagaggccaaggcaggcagatcacctgagcccaggagttcaagaccagcct gggtaacatgatgaaacctcgtctctacaaaaaaatacaaaaaattagccaggcatggtggtgcacacctatagtcccagccacttag gaggctgaggtgggaagatcacttgaggccaggagatggaggctgcagtgagctgtgatcacaccactgtgctccagcctgagtga cagagcaagaccctatctcaaaaaaaaaaaaaaaaaagaaaagctcctgaggtgtagacgccaactctctctagctcgctagtgggtt gcaggaggtgcttacgcatgtttgtttctttgctgccgtcttccagttgctttatctgttcacttgtgccctgactttcaactctgtctcctt cctcttcctacagtactcccctgccctcaacaagatgttttgccaactggccaagacctgccctgtgcagctgtgggttgattccacaccccc gcccggcacccgcgtccgcgccatggccatctacaagcagtcacagcacatgacggaggttgtgaggcgctgcccccaccatgag cgctgctcagatagcgatggtgagcagctggggctggagagacgacagggctggttgcccagggtccccaggcctctgattcctca ctgattgctcttaggtctggcccctcctcagcatcttatccgagtggaaggaaatttgcgtgtggagtatttggatgacagaaacacttttc gacatagtgtggtggtgccctatgagccgcctgaggtctggtttgcaactggggtctctgggaggaggggttaagggtggttgtcagt ggccctccaggtgagcagtaggggggctttctcctgctgcttatttgacctccctataaccccatgagatgtgcaaagtaaatgggttta actattgcacagttgaaaaaactgaagcttacagaggctaagggcctcccctgcttggctgggcgcagtggctcatgcctgtaatccc agcactttgggaggccaaggcaggcggatcacgaggttgggagatcgagaccatcctggctaacggtgaaaccccgtctctactga aaaatacaaaaaaaaattagccgggcgtggtgctgggcacctgtagtcccagctactcgggaggctgaggaaggagaatggcgtg aacctgggcggtggagcttgcagtgagctgagatcacgccactgcactccagcctgggcgacagagcgagattccatctcaaaaaa aaaaaaaaaaggcctcccctgcttgccacaggtctccccaaggcgcactggcctcatcttgggcctgtgttatctcctaggttggctct gactgtaccaccatccactacaactacatgtgtaacagttcctgcatgggcggcatgaaccggaggcccatcctcaccatcatcacact ggaagactccaggtcaggagccacttgccaccctgcacactggcctgctgtgccccagcctctgcttgcctctgacccctgggccca cctcttaccgatttcttccatactactacccatccacctctcatcacatccccggcggggaatctccttactgctcccactcagttttctttt ctctggctttgggacctcttaacctgtggcttctcctccacctacctggagctggagcttaggctccagaaaggacaagggtggttgggag tagatggagcctggttttttaaatgggacaggtaggacctgatttccttactgcctcttgcttctcttttcctatcctgagtagtggtaatc tactgggacggaacagctttgaggtgcgtgtttgtgcctgtcctgggagagaccggcgcacagaggaagagaatctccgcaagaaagg ggagcctcaccacgagctgcccccagggagcactaagcgaggtaagcaagcaggacaagaagcggtggaggagaccaagggt gcagttatgcctcagattcacttttatcacctttccttgcctctttcctagcactgcccaacaacaccagctcctctccccagccaaagaag aaaccactggatggagaatatttcacccttcaggtactaagtcttgggacctcttatcaagtggaaagtttccagtctaacactcaaaatg ccgttttcttcttgactgttttacctgcaattggggcatttgccatcagggggcagtgatgcctcaaagacaatggctcctggttgtagcta actaacttcagaacaccaacttataccataatatatattttaaaggaccagaccagctttcaaaaagaaaattgttaaagagagcatgaaa atggttctatgactttgcctgatacagatgctacttgacttacgatggtgttacttcctgataaactcgtcgtaagttgaaaatattgtaagtt gaaaatggatttaatacacctaatctaaggaacatcatagcttagcctagcctgctttttttttttttttttttggagacagagtctcactct gtcacccaggctggagtgcagtggcgggatctcggctcactgcaacctccgccttctgggttcaagcgattctcctgcctcagcccactga gtagctgggattacaggcacctgccccgacgcccagctaattttttgttatttatttatttttttttttagtagagatgaggtttcaccatgt tggccaggctagtctcgaactcctgaccttgtgatctgcctgccttggcctcccaaagtgctgggattacaggcgtgagccaccgcacccg gcctgcctagcctacttttattttatttttaatggagacagcatcttgctctgttgcccaggctggattacagtgatgtgatcatagctcatta taccctcctgggctcaagcaatccccctaactctgcctccccagtagctaggaccacaggcatacaccaccatacccagctaatttttaa aattttttgtagatagatagagtctcactatgttgcccaggctggtctctagcctacttttttgagacaaggtcttgctctgtcacccaggctg gatagagtgcagtagtgcagtcacagctcactgcagcctccacctcccaggctccatccatcctcccagctcagcctcccaagttgctt caactacaggcctgcaccaccatgcctggctaatttttatttatttatttttattttattttattttatttttttgagactcagtctcactct gtcgcccaggctggagtgcagtggcatgatctcggctcactgcaacctctgcctcctgggttcaagtgattctcctgcctcagcctcccgaat agctaggactacaagcgcctgctaccacgcccagctaatttttgtatttttagtagagacagggtttcaccatgttggccaggctggtctcga acttctgaccatgtgatccgcccgcctcggcctcccaaagtgctgggattacaggtgtgagccaccacgcccggctaatttttatttattta tttaaagacagagtctcactctgtcactcaggctagagtgcagtggcaccatctcagctcactgcagccttgacctccctgggctccgg tgatttcaccctcccaagtagctaggactacaggcacatgccacgacacccagctaattttttattttctgtgaagtcaaggtcttgctacg ttgcccatgctggtatcaaacccctgggctcaatcaatccttccacctcagcctccccaagtattggggttacaggcatgagctaccaca ctcagccctagcctacttgaaacgtgttcagagcatttaagttaccctacagttgggcaaagtcatctaacacaaagccctttttatagtaa taaaatgttgtatatctcatgtgatttattgaatattgttactgaaagtgagaaacagcatggttgcatgaaaggaggcacagtcgagcca ggcacagcctgggcgcagagcgagactcaaaaaaagaaaaggccaggcgcactggctcacgcctgtaatcccagcatttcggga ggctgaggcgggtggatcacctgaggtcaggagttcaagaccagcctagccaacatggtgaaaccccgtctctactaaaatacaaaa attaaccgggcgtgatggcaggtgcctgtaatcccagctacttgggaggctgaggcaggagaatcgcttgaaccaggaggcggag gttgcagggagccaagatggcgccactgcactccagcctgggcgatagagtgagactccgtctcagaaaaaaaagaaaagaaacg aggcacagtcgcatgcacatgtagtcccagttacttgagaggctaaggcaggaggatctcttgagcccaagagtttgagtccagcctg aacaacatagcaagacatcatctctaaaatttaaaaaagggccgggcacagtggctcacacctgtaatcccagcactttgggaggtgg aggtgggtagatcacctgacgtcaggagttggaaaccagcctggctaacatggtgaagccccatctctactaaaaacacaaaaattag ccaggtgtggtagcacacgcctgtagtcccagctactcgggaggctgaggcacaagaatcacttgaaccccagaggcggagattgc aatcagccaagattgcaccattgcactcccgcctgggcaacagagtgagaccccatctcaaaataaataaataaatatttttaaaagtca gctgtataggtacttgaagtgcagtttctactaaatgcatgttgcttttgtaccgtcataaagtcaaacaattgtaacttgaaccatctttt aactcaggtactgtgtatatacttacttctccccctcctctgttgctgcagatccgtgggcgtgagcgcttcgagatgttccgagagctgaat gaggccttggaactcaaggatgcccaggctgggaaggagccaggggggagcagggctcactccaggtgagtgacctcagcccctt cctggccctactcccctgccttcctaggttggaaagccataggattccattctcatcctgccttcatggtcaaaggcagctgaccccatct cattgggtcccagccctgcacagacatttttttagtcttcctccggttgaatcctataaccacattcttgcctcagtgtatccacagaacatc caaacccagggacgagtgtggatacttctttgccattctccgcaactcccagcccagagctggagggtctcaaggaggggcctaata attgtgtaatactgaatacagccagagtttcaggtcatatactcagccctgccatgcaccggcaggtcctaggtgacccccgtcaaact cagtttccttatatataaaatggggtaagggggccgggcgcagtggctcacgaatcccacactctgggaggccaaggcgagtggatc acctgaggtcgggagtttgagcccagcctgaccaacatggagaaaccccatctctactaaaaatacaaaagtagccgggcgtggtga tgcatgcctgtaatcccagctacctactcgggaggctgaggcaggagaatcgcttgaacccgggaggcagaggttgcggtgagctg agatctcaccattacactccagcctgggcaacaagagtgaaactccgtctcaaaaaagataaataaagtaaaatggggtaagggaag attacgagactaatacacactaatactctgaggtgctcagtaaacatatttgcatggggtgtggccaccatcttgatttgaattcccgttgt cccagccttaggcccttcaaagcattggtcagggaaaaggggcacagaccctctcactcatgtgatgtcatctctcctccctgcttctgt ctcctacagccacctgaagtccaaaaagggtcagtctacctcccgccataaaaaactcatgttcaagacagaagggcctgactcaga ctgacattctccacttcttgttccccactgacagcctcccacccccatctctccctcccctgccattttgggttttgggtctttgaacccttgc ttgcaataggtgtgcgtcagaagcacccaggacttccatttgctttgtcccggggctccactgaacaagttggcctgcactggtgttttgt tgtggggaggaggatggggagtaggacataccagcttagattttaaggtttttactgtgagggatgtttgggagatgtaagaaatgttctt gcagttaagggttagtttacaatcagccacattctaggtaggggcccacttcaccgtactaaccagggaagctgtccctcactgttgaat tttctctaacttcaaggcccatatctgtgaaatgctggcatttgcacctacctcacagagtgcattgtgagggttaatgaaataatgtacat ctggccttgaaaccaccttttattacatggggtctagaacttgacccccttgagggtgcttgttccctctccctgttggtcggtgggttggt agtttctacagttgggcagctggttaggtagagggagttgtcaagtctctgctggcccagccaaaccctgtctgacaacctcttggtgaa ccttagtacctaaaaggaaatctcaccccatcccacaccctggaggatttcatctcttgtatatgatgatctggatccaccaagacttgtttt atgctcagggtcaatttcttttttcttttttttttttttttttctttttctttgagactgggtctcgctttgttgcccaggctggagtggagt ggcgtgatcttggcttactgcagcctttgcctccccggctcgagcagtcctgcctcagcctccggagtagctgggaccacaggttcatgcca ccatggccagccaacttttgcatgttttgtagagatggggtctcacagtgttgcccaggctggtctcaaactcctgggctcaggcgatccacct gtctcagcctcccagagtgctgggattacaattgtgagccaccacgtccagctggaagggtcaacatcttttacattctgcaagcacatc tgcattttcaccccacccttcccctccttctccctttttatatcccatttttatatcgatctcttattttacaataaaactttgctgccacct gtgtgtctgaggggtgaacgccagtgcaggctactggggtcagcaggtgcaggggtgagtgaggaggtgctgggaagcagccacctgagt ctgcaatgagtgtgggctggggggcccagtgcccgggttccgggaggggaacaaaggctggagactgggtcagtctgcgggctg catgacaacaagggagggggtggctccattcataactcaggaaccaaccgtccctcctcccctccggccacggctggcacaaggtt ctctccctcccctgcttctaggactgggctgcttccccctcggcagcctctcaccaaggattacgggatttaaatgtctgatttagcaagg ctgagcctccagggtggccatctgctccatcagaaagtggcaggatacctgggttcccaaggggaacaggggtgggtgctactgga tggagagaggccagtgggaggcctgctagccagggtcccaggaaagtgggggcagctaaggtaagagtaggggtgtggggcta ggtccttcccagcatcccctcatcctgggcctcatgccaggtagctgaatgaattgaagctttaaactctgccaggaaaacctttcaaag ggcttcttgggatagggaggagagtcgggttgaggagctcagtactgcctgcccatgctcctcagggctgctggctcccagggagg ggggctgggagcaggcaggctcttccccatcacccactgctctcttggagccagtgcttgaaggggcagtcagacatggcttgccct tcctcctccctggtggtggagatgggtgttagggtccagtgggtgctactgtccaggggggcttctggggccaccagcctgtcagctc atcaaccaggctgaaggtgcaagcaggagccccttgccttgccccaaggatcccagacagctatgaagccaccagccttcctgacct caagaccaccttttttttttctctttcttactagggaatgccaaacactctccccaggagatccagacccgcctctttcagagacttttaactt aaacatctgtccctacccagcaggcaaactagagctcctgaagctcagtccctgtccttgcctctgtagacaggtcaccttgatgagctt cctttttttttttttaatttttttttattttaggctttattggggcataattgatcccccaaaattgcatacattcaaggtatgcagtgtgatg atttgatatgggggtatattgtgaaaccattaccacaatcaaattaatcagcacgtccatcatcacacacagttaccatttgtgtgtgtgcac gtgtgttcacctacgacgaggacacttggacctactctgcagatctcaagtaaacagaaaatctccctttttgacaaccatcctccacccttt caatcccaaccttttcctagattatgtccctagctctgtttttatttctgctgtgctgcttcagatccattctgactctgccaaacccttctt tgtgagctgatagattgctggattgagaattacagctgggcgcggtggctcacgcctgtaatcccaacactgtgggaggccaaggccggcggat cacttgaggtcaggagttggagaccagcctgaccaacaagatgaaaccccatctctactaaaaatacaaaattagctgggcatggtgg tgcacgcctgtaatctcatcttcttgggaggctgaggcaggagaattgcttgaacccgggaggtggaggttgcagtgagccaagatcc tgccattgcactccagcctgggcaacaacagtgaagctccatctcaaaacacacaaaaaaaagaagtacaaagtctgagacttcagg ccagctctgctacactatatactctaacctctctggtcctacttggtgacttctttccctctggtcgtgttcaagttcccgtcccatccagtca agcaggtactcattggtaccttaccctgtgccaggagctgttctaggccctggaaacctatggcagacatgttccctaccctcccactca aagagcccaggccttatcctaatgagatctgaaatcaaatctcccaatttcctcatggcttcagtctaaacttgtaattcacaaccttaaatc aatatgttctatttttttatttagaaaacatttccggccaggcacggtggatcacacctgtaatcccagctactcgggaggctgaggcagg agaatcgcttgaacccaggaggcagagggttgcagtgagccgagattgcgccattgcactctagcctgggcaacagagcaagactc catctcaaaaaagaaaaaaaaatggaagaaaaaaaaatttccccctcattttaggaacacgaggtctccaaatctaaaattcgtactctg aggagattgaatagccttaaatgctttcatcattaaaaagaaaagaaaggaacctggtatgcatcctaaaaatgaaaaatatacctacct gtaatcccagcacacagcacattgggaggctaaagcaggaggataacttgaggccaggagtttcagatcagcctgggcaacatagc aacaccccatttctttttcttttcttttttttttggagacacagtctcgctctgttactcaggctggagtgcagtggctcaatctcagctcac tgcaagctctgcctcccaggttcatgccattctcctgcctcagcctcccgagtagctgggactacaggcgcccgccaccacgcctggctaa ttttttgtatttttagtagagacagggtttcaccgtgttagccaggatggtctcgatctcctgacctcgtgatccgccagccttggcctccta aagtactgggattacaggcgtgagccactgcgcctggccacaacaccccatttctattttaataaaataaaatactgtgaaaaacatttac aatttttaaattttaattttaaaattaaacttatatttattcatttgtgtgtgtgggtttttttttttttttttttttgctttttttttgaga tggagtgtcactctgtcacccaggctggagtgcagtggcgtgatctctgcctcccggttcaagtgattctcctgccatagcctcccaagtagc tgggactacaggtacacgccaccacgccgggttaatttttgtatttttagtagagacaggatttcactgtgtcgccaggctagcctcgaact cctgacctcaggtgattcgcccaccttggcctcccaaagtgctgtgattacaagcgtgagccaccgtgcccagcccaaagttggttttaatag cagaaaatctatcaacataattcaatatattaaatttagaaagaaaaattatctatcatatcaacagatactgaaaggaatttgattaaattt cagtagccatttccttaaaaaagaaaacactttaacacagtaatagactgataatggaataccaattttcctaataagttaaacattaagata atttcaattaaggtcaagagctgggccaggtgcagtggctcacacctgtaatcccaacactttggaggccaaggtgggtggatcacctgagg tcaggagtggagaccagcctggctgacaatagtgaaatcctgcctctactaaaaacacaaaaaattagctgggcatggtggtgggca cctataatcccagctactgggaaggctgagacaggagaattgcttgaacctgggaggcggaggttgcagtgagcaaagatcacacc attgcactccagcctgggcgacagagccagagtcagtctcaaaaaaaaaaagaggtggccacacctataatccaaacattttgtgag gccaaggcaggagaattgcttcaggccaagagttgaacacctcgtcaacatagccagacctctctctagatagatagatagatgatag atagagagatagatagatgatagatagagagatagatagatgatagatagatagatagatagatagatagatagatagatagatagata gatagatagataatctggccgggtgtggaggctcacgcctgtaatcccagcactttgggaggctgaggcgggcagatcacgaggac aagagattgaaaccatcctggctaacaaggtgaaaccccgtctctactaaaaatacaaaaaattaggcgggtgtggtggcacgcgcct gtagtcctagctattcaggaggctgagacaggagaattgcttgaatccgaaaggcggaggttgcagcgagccgagatcgtgccact gcactccagcctgggtgacagagcaagactccatctcaaaataaataaataaataatcaagaacagtataaggggctgtatggtggct catgcctgtgatcccagcactttgggaggccaaggtgggaggatcccttgagaccagcccaggcaacagagaaagaccctgtctct atttaaaaaaattaaaaactggccgggcacggtggctcacgcctgtaattccagcgcttgggaggccaaggcaggcacatcaggag gtcaggagttcgagaccagcctggccaacgtggtgaaaccccgtctctactaaaaatacaaaaagtagctaggcgtggtggcaggc acctgtaatcccagctacttgggaggctgaggcaggagaatcgcttgaacccaggaggcggaggttgcagtgggcaaagatcgtgc cattgcactcagcctgggtgacagggcaagactccatctcaaaataaataaacaaagtaattaattaattaaattaaaaactgtggggat atagacttactctggttttattttttcttttcttttcttttcttttttctgagacggagtctcgctctgttgcccaggctggagtacagtgg cgtggtttctgttctctgcaacctccacctcccggattcaagcgattctcttgcctcagcctcttgaatacctggaattacaggtgcctgcc accacccccggctaattttttgtatttttagtagagacagggtttcaccatgttggccaagctggtctcgaactcctgacctcatgatccac ccgcctctgcctcccaaagcactgagactacaggagtgagccactgtgcccagcctactctggttttagtgcattcaagaggaacaaaaaagga agaaaatcactagtaaatatacctctttctggttagagtggatgtttggaaattatatatatattatattatattatatatattatatatata cacaaacacgtacatacatgcacacacatatatgcctttttgattataggatagtataccaaaactcagaaatattatggaattaacagaatt tagtaaggcagataagtagtaggtagaaaaatattaattttatcttccagcagaagcactgtgaaaaattagacaacaagaaaacattccattc aaaataatgacaataaggccgggcatggtggctcacacctgtaatcccagcactttgggaggctgaggcaggaggatcatctgaggt caagtttgagatcagcctggccaacatggtgacaccctgtctctactgaaaatacaaaaatcagccagctatggtagtgtaagcctgta attccagctactcgggaggtcgaagcagaagaatcacttgaacccaggaggcagagattgcagtgagccaagatcctgccagtgctt tccagcctgggcaacagtgtgaggctccatctcaaaaaaaaaaaaaaaaaaaagacaatagcaataaacattaagaaatgtgtaatag gaatggcacacacaaagaaggaatggcacagagcctgtatgcagaagaccacaaacccttatttaacgacgtaagccaagatccaa agaaaatgatagattctcagatgggaaaactaaaaaaataagaaaaatcaattatctcgagataaatataatataatgcaatttcaattag aatcccaaattttcattgtgtgtgtgtgtgagttgggtaaatttatcataaatgtataggaacgagtaagtgtcactagttgtttaaataaata ctggatttgggccaggcatggtggctcacgcctctaatcccagcactttgggagaccgaggcgggcagatcatgaggtcaggagat cgagaccatctggccaacatagtgaaaactcgtctctactaaagatacaaaaaattagctgggcatggtggcacgtgcctgtagttcca gctactctggaggctgaggcaggagagttgcttgaacccgggaggtggaggttgcaatgagccgagatcctgtcactgcactccac cctggcgacaaagtgagactccgtctctctctctctctttaggccaaggcaggtggatcacctgaggtcaggagttcaagacagcctg gccaacatagcgaaatcccatctctactaaaaatacaaaaattagcctggcagtggtggcccacgcctgtaatcccagctactaaggg ggctgaggcaggaggatctcttaaccagggaggaggaggttgcagtgagcagagattgtgccactgcactccagcctgtgcaacag agtgagactctgtctcaaaaaaaataaataaacaaaatactggaggccgggcacggtggctcccgcctgtaatcccagcactttggga ggccaaggcgggtgaatcgctttcagctcaggagttccggaccagtctgggcaacatggcaaaaccccgtctatactaaaaatacaa aacttagccaggcgtggtagtgcatacctataatcccagctactcgagaggctgaggcaggagaatcccttgaaaccgggaggcag aggttgcagtaagctgaaatcgtgccactgcactccagcctggacgacacagcgggagactgtctcaataaataaaataaataatata aaataacataaataataaaattgtaaataataagtaaataataagcaacagaatggagagggggtcctatttgccttgccagattttagag aacttagtataggctaggcaggtgcagtggctcacgcctgtaatcccagcactttgggagtccaaggcagtggatcacatgaggtcag gagttcaagaccagcctgaccaacatggtgaaaccccatctctactaaaaatacaaaattagccaggcatggtggcacatgcctgtaa Protein: SEQ ID MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTED NO: 347 PGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFL HSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHM TEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVG SDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRT EEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFR ELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD CDKN2A SEQ ID gcccgcgaggtttaggacggatccaggcagaccgcaggctccgggtcggggcaccgggtcagcgcgccggcctgaaggcggc NO: 308 gtcctgggctcgacttcccgcgcgcggagagccggcgagcccgcgtccgagttcctggacgagagccgagcctcgcttagaccgc gctcaggacccggctcctccgcattctccggctgcccctgtgtcctcgactcacccctcctttctgccgctccttcctttccttgccctgct tttactgttcccaaacaggaccgcttttcctgtctcccagctggaaaggaggaagggagagagtccagaaaggatcggtgatgtggaa gaaaaggggaggaggggacatggagggggagaccggagagagaacgtacgccgaggagtcaggcggcgggatcaagggga gtcggggtgtctgggcgcggggcagagcgtggaggcggcagcggccaacggtcgccaagacaaccattctacgcgaggacgcg gcgacaggaggggagcggccagcaggggaggggagcgcgggggaagaggaaagaggaagaagcgctcagatgctccgcgg ctgtcgtgaaggttaaaaccgaaaataaaaatgggctagacacaaaggactcggtgcttgtcccagccaggcgccctcggcgacgc gggcagctgggaggggaatgggcgcccggacccagctgggacccccgggtgcgactccacctacctagtccggcgccaggccg ggtcgacagctccggcagcgccagcgccgcgccgtgtccagatgtcgcgtcagaggcgtgcagcggtttagtttaatttcgcttgtttt ccaaatctagaagaggagcggagcggcttttagttcaaaactgacattcagcctcctgattggcggatagagcaatgagatgacctcg ctttcctttcttcctttttcatttttaaataatctagtttgaagaatggaagactttcgacgaggggagccaggaataaaataaggggaatag gggagcggggacgcgagcagcaccagaatccgcgggagcgcggctgttcctggtagggccgtgtcaggtgacggatgtagctag ggggcgagctgcctggagttgcgttccaggcgtccggcccctgggccgtcaccgcggggcgcccgcgctgagggtgggaagatg gtggtgggggtgggggcgcacacagggcgggaaagtggcggtaggcgggagggagaggaacgcgggccctgagccgcccgc gcgcgcgcctccctacgggcgcctccggcagcccttcccgcgtgcgcagggctcagagccgttccgagatcttggaggtccgggt gggagtgggggtggggtgggggtgggggtgaaggtggggggcgggcgcgctcagggaaggcgggtgcgcgcctgcggggc ggagatgggcagggggcggtgcgtgggtcccagtctgcagttaagggggcaggagtggcgctgctcacctctggtgccaaaggg cggcgcagcggctgccgagctcggccctggaggcggcgagaacatggtgcgcaggttcttggtgaccctccggattcggcgcgc gtgcggcccgccgcgagtgagggttttcgtggttcacatcccgcggctcacgggggagtgggcagcgccaggggcgcccgccgc tgtggccctcgtgctgatgctactgaggagccagcgtctagggcagcagccgcttcctagaagaccaggtaggaaaggccctcgaa aagtccggggcgcattcggcacttgttttgtttggtgtgatttcgtaaacagataattcgtctctagcccaggctaggaggaggaggaga taaccgccggtggaggcttccccattcgggttacaacgacttagacatgtggttctcgcagtaccattgaacctggacctcccttcacac agcccctcaatcgtgggaaactgaggcgaacagagcttctaaacccacctcagaagtcagtgagtcccgaatatcctgggtgggaat gactaagacacacacacacacacacacacacacacacacacacacacacacacagtaggaaaggtgtatttcaagcacactttctttc tccttggggagaattattgctaaccatctaagttttctggaggcggccttttttctccccagcctcccggcggggtcaccctctcccacctt ccaggagagtggaggacccgtgagatacggggcacgcaggcagcgacttcctgaaatgctaacaaggatcgtaggatcagttact gctgcgaggagcaagcacttgcttcttgggggagttttgcagccaacagggaaatgggctttctttgtgagttagaggtagaggtccg gcggcctgagtgattgaaactgctcgggacaatgctcgtatgtttagcaaacgacagaactgtagaactgttcctgagaaatcccaact gatagtattttagtcatctcagacgacagttagcacagtttaaaaatgaggcctacttcttgaaaaacagaatccaaggtagttttgtcctc acattgacaaatgttgacacagccagtgtaatttcctataaccaggaaaactgaaagaatatatgtacagttaaaatatgtacaatgctaa ttaaaacttgtgtaataagtctaaaagtaatttaatgaggcttcacttttatgaccgtccttgtggtatgcttcgccaggaatatatagcttca aaaagcaaaggccagcggaggggtaattatttttttactgcaatgttaattgtctctttgacatggaaatataaacctgttaaaactatcagt gtttaatttagtgtctcaatttctattagcaaaaatttataatctataggataaatgcacattttattttttacttttcatattatgcaagt taatttttttaatttagtcaaaggagcttataaaggatttcagggcctgttgctggatttgattttaattcattttgaaacattgacaagac cctggttgttgttttttttaacagtggtttatccgtatcagcaaaagtttagccactgtgaccggtaactgtatgaatatagttcttaatat tattgtctatataaaaatatttattactctagttaatattattctatataaaatcattttgtttaaattattaagttgcctctgaaaatctg tagtaacaaagtagaacatgtcaatgtatataaatgccataattatgtattttttagtttaggcctataaaacataacattgtggtgatttt aagttagagaaaatattttatagtatgttaatgtatatgcatgaaatgcaaaaatatttaaatgataggttcattgaaatagatcatttttt gttatttaggtataaatcaattttcaggacgtatgtgaaaagcgcaatcttcaggaagtttctcaagatagaacacagcttggatagaatgt cttgaaatatatgcaattttccaatttcatatgtaaaatgatatacataatataaaatctagcggtgttaattataatgatatgtaattata tatttcacattaatatattttatgcccatggctatattgatttgggaatatatatggatactaattatgttaggattcatacaattccttga gaggcacaagtgctaaaaattacttgtatgaattatttaatatcattgcaaataagatgttattttaactttttttaagtttctgcaaatat gtttattatgactttttatttttatatgattggaaatacatatactaaaattccacgttaccagtttcttaaccacagaaacctgaaaaatt gccatagttgatttgttacttctaccttggtgcatttacaaaatagtcatatttttattatgaagttaaatattcatttgtttatagctact tcagaaggctcaggttatttttttctttaatagcacagagtcctctcaaggtaagcactgtgcagttagtataaaccattattccccatgtg tacatgattcacagtttgtattgtgttccaagtgaaccatagccctttcagaaatcaagacttatattcattttacttctttgagtactctt gaattttagaaagtccattatgatcctaaggtagcaacaacatagcctattaccgtctatgatggtttacagatctattattccacgttagt tcatcactatcaactaccatgatagagttaagctaaaccattttcccaacatatgaaaaactcctattactaaagtgatacaaatggtatc aaaaatacttttttatagcaaggttcaacagtgggcccagtgcttttacactttttcaaaagtccttggagaaacagagaaaatctcacttg ccttctgtactaaaacattctaggccgaactaaaactgaaacttcatagtagaacactgtaggccaggggtgttcaatcttttaccttccc tgggccacataggaagaagaagaattgtcttgggacacacattaaatacactaacactaacaatagctgatgagcttaaaaaaaaaaaaaaa ctcatgatactttaagaaagtgtatgaatttgtgttgggcagcattcaaaaccatcctgggctgcatgtgaccctcgggcccacaggttgga caagcttgccctagctcctccatctgctgcaaagcccagcctgatacaaaaaccaacgtgataaaaagtttttgtggtgctttattttggc agtttaagttatataaacaatgggtacagtttcattttctaaatataaaatttttacattgaatatgaatttttaagacaaattatctgaa ttctgattctcatatacctaactactaatatcttctctatttgttgcccaatgagattaatccacctcttaaacacttcaccatcaagaaa aacaattttgtattttaaaatgaacccatccactttcattcagctattttatattcaggcatcatcctaaggaa agaaaggttctgacaaagattaatacagatggataagtagtagcaagaaatcaaaaactgcataaaattctagcaataaagtgttaaatt atggtacagttacattctggatcatcaggtatctgaagaatatttcatagactgttaaatgattgcattataaagtcaggtttttttaaagca agattccaaacagtaaacagtttctctctctctctctctctctctctctctctctctctctcaccaaattagttataatggtttccgcagga tgagaggggttgggaaaaagtttggtgatatttattttcttcgtttcacttttgagttttccaaagtgctatgaccatcatcagtaaaatat acatttccaaagcctttgacacacggtaacagtcctacacagtggatgaactaagagcttctctacccttagatgggtagggagggaggaaa gacaaggaaactgagttgtttaagtgtcatacacgagaacgtggctttaaggtctgggaaaacctgcgagggctgtgacgtcagactgtgaa atgcacgctatgtccattcaccaagacgttccattttaaaacccataaatccgtagctatacctgtttccaaggtgcctcgtgttaggcctc tggtcacagcacttggcgcccttcttgggatctcttctctccgcccccactaccccaccccacaagcacactctagtcccctccaatcaa tttcaggcaggtctcgccgcctccggagccacgctgggggtgcaagggccctggacccgaaagagcgcccgcccggcgacaag agatgagatgcacgctgctcctccactcctcagcccccaccatcctcctcctggatcctaacttccccactctctcaattcctagagacg ctgcggatcccagaggcttaactggcagctggaacgaggtcctccaacaagaatttagacgctaggtccaattatcactccaccgcgc gcactttccgcaggagcgatgtgatccgttatcataactgcggacctggggttccacgtggaagacgattgggatttcactggccgcg gtgggggtgggagcagacagagtctgagtggggttagtggactcgagacgaaaggcaggacatgacagaaggcaactctgggtc acctctccagcttggaactggctaggccttgttttggaggggatgggtagatgaaaagtgagtcagggttacccggaggaaccacgg ggaaagtgcgcttctgagactcttgacagccatttcgttcccttccaagccagatggagacccaagagtgttgaaaggccacgacttcc ctcagtttctccatctgggggtgcaggatggtatagagagtggcccgtagtatttttccagtgacgatgtctctccattgttttcttcttata ttgcagctttccccatgtttgaaaattttcttttcaaatgaaatcattgattagaataaaaaaaagtaagtagctattaaaacaagatcaat ttccatgacagtaagccaaccgatggagaaaaccttgggaattaataaatgaaggatttgtttggtagatgataaaaggtccttttaaagggtc tgactcttcctagaaaaacccaccaacttgggaccgcaacagatttaccatatcctaattcatgctattttaatgtgtattcagcaaaccca catgtgtttacaattgtcgaagctaccaaatgtcaatagcgttttttttctatttgttgaatgtgaatctcttgtacgaagccatataaacaga agaaattacaggaatgattttaaatcacatacaaaaccaatagtattgctagaggagagttagtcaaggacggcattatgaagaaagtga gggagaatttccaaagagcagaacgatagggcttggtggaccaaagaacgtttccatctaaagggaatggcaaatacttagagtctct gaacccactgaatcttggactatttaactaatatttgtagttccagatatagcacagtgccttgtacatagtggtatttttaaaaatatagtgc ctcgtagattttttttcaacttttatttaggaggagagggcacatgtgcaggttaattacaaaggtatattgcaccatgctgaggtttcgagt acgactgaatctgtcactcaagtagtgagcacagtacccacagtaggtagtatttcagccctcgctcattccctttctcctccatctagtag tccccaatgtctattgttctcatatttatgtccaattagcatttgttttttaaaaagggtggttgaagaaattctcagtgcttgtcagtgtct ctcagtgcattcatttaattcatgagccctggaatgatggtttcatttgggcagaactctacaatcaaaaagaagtaataaaagggaaaaaaaa gtgaaagccatcaactacaggattgaaattcccaaagcatcagaggtcctttcaaaaaatagtatgttgatttttaatttttatgacttattgg ctttgttcatgaaaatataaacatgttatcacaaaggattttttaattcaactatttctcagttttctctttcaccttcaaaataaaatatca taaattatttaaatggttgtgaaggcagtaggatttttttaagagagaaaagttttatagaggttcagaattacatgaacaaagacatgtaat ctcttaagcaaattgaaactaataaaatcgtacaatcaaggtaacgtaaataaaaaagcctctgctttcttaattgaattatgtgagtaacta gaaattttaaaagtatggcaaaggttaacaacagcattattacctgggctgcctttaaaaatacatatttctggggttcacgttcagaaaat ttgattcagatttgctgtgggtcccagaaatctgcattttaaataaacacttgaaggagatactaatacaagtggcccattgggacacaatt tgacaaatatgaccaattttactttttaaaccttatttctgcttctttatctttgaattgaggtccaggattttaggtaagattttaagttt agagtcagtttactggatcccagggaggagagtctgagtaatcagtggaggagttatttcaccaaatgaaggagaccctttattattatgtga ccctttgtatgaattggaaaagaatgtcttgtagataccacatttttacagtcagaacatagtttgagagaaaaaaatataacaagatatat ttgtgttttaaagcttacagaaccagacagaaaatttccacataagctatataagatacgttgtctttttaaaacactatatacacttctttc tgttcgtgcaggatgaatggatctctctctctctctctctctctctgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgttgtaataagg ggtttctttcattttatgatccagaccaggctcgtaataaacatgacaacctaaaattatgtaaaaaagaaaaatcaaagcacaagtgtttc acaggtttaacttatgcttatctaagatcagggcaagattgcaggaaaatgtagccataacagaataaagcatttatggacaaaatgatggg ttctttatgtctctgaaaagcacagtgatggggggggaaatatagatgaaaaatgtaagctaaaaagtaacaattataagaaaaactaaaat atcatgcctttcaaatgatcatttttctgcttttaagctaaaatttgtctaatattacaccagtgactttgctgatgtattaggaaaaagct tgttttgctttcttttctcgagtgccaccattttcttgctctcattctctttcaggctgccagatcatctgactcagcaattgtataactct ctcacccaatttaaagaaacagcagctgtctagagaacaatgactcccccagttgaacatctaattgttaaatgtccaacatcggacacttt gaattttactccatgcaatttacatgctgaatagttgaagttgaatatattatatttaacatttaatttttaaaagcttattgaaactttc ttcctaaatcacatggtaaagttattgttttcttcaaaaacaattaggaggagcttaacaataataggacacttcaacttccattatctaat ttaattatcacaatatccttatgttttcaatgtttcattttttcattttgtagatctggagactgaggctcagataggttgcatggcctacc aaaagtcattgactagtaattcatatatagttgaacttggttgcccatggagtgctataaatatgtatatggtttcagttccatctcttttag ttaactattattttgaaagtcgcttaacccctttgggcctctactatactcaagcatcagccgtataagtcacagtaaatatttattggttg aaaggaggttaacatctttcaaaaatttattttttgaccaaaataaaaccagtgaaaaattctcatatgactgtacatataaattacttatt cctaccttaatttaaaagcaataagtgggatacctattcaccagcacaggaaccacttgaagcgtgcagttgaaagattactttctttagca ttcacatgacctgtgagcagattctatttcttttgcttattagctgtcatggtaccagaatgaagtatgagaaactctcagtgctttcatgt tctcatctgtaaacctgagaccctatggtagtcccgtaataagaggtagataaaatagtatgtgtgaagagtcactgtaaacttttacacag tgtacgtttgtcagttattatagtgcctaattaaactatgcccttaagaaagcacattagttttttacagtaaatacctacttcattata atttttcagtgtagctagaaatttctaaactccactttaaaaatatacatatcataataaaaatatatttatgtattcagactcctggtat gttccaaggtgttaggtaaaatcagtgtaaatttgcatacttttaaattcacatctgtacagaagatctatatggtggcctttagggtatac ctctaagctattctagtattcataatcattaaagagatattaagcagtgtttgtgaacccctgttttctaagacaggaaatcaaggtagctt tagaaaactggaaaaaaagttattagtctatctatctaataacccagaataataatttccaaaggaatcactgaagataactggatttttaa ttccttcagaatggttgtcacagtctgaatatctgaatcaacagttttgaccaaaacaattttctaaaaattctttagtataaaaaattat gtgtgtgtgtgtctgtgtgatgaaaggaatgataggcagaaacattactgtcatccttacgacattcaaaatgcctaccttggagggtgac cttcagttatttttatgcaaatgtgaagaagttatttagaagtaggatatcaaagagtaacacaaaatacactaaatagtatgctttctta aggctaaattgacttgggggttttaaatcagtacagagtaaacatacagtatattctgttatcattgcctttttgaaaaattaattatgg aagttatcatcttaaccgtaacaacacaaaagataaaactctaccctcaacccagagactcaaaggaaaacatgagtggaaatgttaaat ctgtatgtgaaaagtgctaaaacatgaataggaagcagttacttatttaatcaaagttgattatatttcatcaagaagttgattcccttga gtggagttgaatcacatatcaggtgaagaatgtgatttggggaagaatggtctaacacaagaaaattttcttgcaatctttaataatatca gaggggagattggcttcagaactctcctaagttcaggaaaggacacagaaaattgaacataacagtaagactatagagtcccaagaaagca agctacttttaaaggatagttttttagaggggcaaaagggggacaaccattctccatttgatgagaaaagcttccatgtagatggtgcccc tgaaattagagtatcctaaaccagtgttaaacctatcagtgaaacatgaatattaaacctccactcccagtagtgaaaaccgaatacatta ttatttatctgtgactttcaacattatctcagaactctaacagcacatgcgtacatcagcagcataagcagaaatgagatattatatatgc ttgtgttagcaattaaaaaggacagcatatttgagaggggaaaatctgtcctatcaagaatgaaaaagagggaggttaggaaaagtagttt agagaaagtaaattttgcaattcctcagttttaactgtagtttctccattgtaccttccacttgaaatgcactccaagcagtggaggtggg tagcaatgaatgcagaggaaacactgaacacagtgacactctccagtgtcacttctcatgatttaatgaggggttttttttggaaattctt ctgtcataacatgggaaactttgttacaaagaagctgttttttcagagggttagaattcagaggtagcatcataccttttagaagagaat ttgcttgttgaaaccacagatacctgctagaatgtacaggaattaatgaaaaattactcaaaaggacatttattttgatgacctaaatgaa taacttcatagtaaatgtcatatatattctcaaaaaattaaaaagcaccatttattgagagcctaccgtgcacccggatttttatatatct gacattctttattcctcacagtaaccttatggggtaaattttattttccccactttgtgaggtgaggaaataaaggctcagaaagtttac ataacttattcaagcccacagagctggtaaatgagaggtcagttctatctgagtttaaagactaggcttgtcccacttgcatatgtgtca tttccaaaattatgattaaggatatggttggcatttcccgccacccacattaagtccaattaagtagctgtggccatagaaagaatggaga atggagagaggaactgacttcaacagctacagcaaacatttattagctgagtaaccatagctacatagttcctcaatatgtaccactcct ccattttgttatctataaatcaaaatggtggctttttaaaaagcagttttacaatatattcaagagccttctaccctttgaaaaactgcaa tactatttttagtagcaattagaaacaccttaaatatctgacaacagggacatcattaagtaaattataactttttccagtgggatgtgtt acagctgttaaaagtagcatttatgaagtgtttttggagaagtttggaaaatgctgtaataagttagaaaaagctcatttcaaaattgca taatattcacaatgtaaagattaagcaaagaaaaaggaagaagtatttcaaaatgttaataattattgctttgtgtggggtagtttttcatt ttctatgtgcagctaattccttaattatttttaaatatgtgagctttaatcaggaaagcaaatcattcaaaaatgaggggactgaattaag tgactttcaggggactttgcgtgtctttgagttccaaatttctatcactatgtattactactgaagaataatcatagaagcacagtagttt ctgaaaatggagagtcagtaatcttggcccaggttttgcaacttgctctaaagcagagtcctcaaagaaaaggaagcattgatgagttgtc cacaatgtactggataaattatcattaggaaaacatattgtagtagggagagtgaggacctctcaaacagaactgagaaccttaagtttg aacttttcttttccttattacttaagcactctgagcttttttttttgtctgcattatgaagaaagaataatactctctatcccatgggaca gctgtggaattataaattacacatataaaactgcttgatgcttgtcacatagctgggggttgaaaaaatgatagccattattttcttggca acttttaatgaattttttattatctctatttctttctgcctatctcctctaattatgtttattacttattttgttcctcaggatgaggtc aattctcaatatctgtgctgtacataatatacatatataccaaatatgtgcatatagtatgtacatacatacatactgtgctaatctttt agtgttctcagctgatcaaatagctacaaatagatataagtaattcgccacaagtaatttatcaacataaaaaaaatttacaaaaaagtt aaggaataattgtctccatgagctgcaaagatccctcatttcacaagagtacaccctagagatattttaatagtaaatttctcacatagat ttaaaatcacatttgttttgcacataatttagaaaagatacctgctatataataagtaatatacttttaagtttccttcaaaatattctt gggaagatgataataggtactgctaattctatacccagttaacattttggaaactaaggttgaaaattgtgacttaactataattatgcat taaatctacaacacatcaaagaattttgcattttgtactccttactaagatccagtttgagtaggaagataaattttacagtaattctgaa tgagggaagttggcacagagtttctaaaagagtaccttccttatagcaaatactaaataattgtgctatattgaatttaattaaatagaga atagtaaaagggagaaagaaacatccaatgttttgaaacttctagagatctactcccagggacacattgttttttcttagcaaatctgtt ctggaggtctgctctactttctcagaggtctccctttcatgctgaagctatcttttttcttgtggaacataagtaattaaataccttgca attatttacctaagaaagtgtttctttcccgtttaaaatgctcttaccacccacattggactcgattatcagaatttttatccggggcag cttcaggagcactttggcacttcggggctaaaccacaatctgtttttacatgtttgtgattatacccgttttgtagatcaagacattgaa ggctagtaaaaaaaaaaaaaagtcattttttcagggtaacaaagtaggtggtagaactaggacaggactctaatttccttacattattgc ttttctaaattaaagggatgcatggaattattcctccattgcctttgccttcaaataattatctattgcacccaacatcctattctagaa ctcatctatgaaggcttaacacagctgtacctgggagctccattacagggcatatatctcgctctcataagctacttcctaaggaattct ctttaattatgggagcttttccagactctgaaatctttttttcctggtaacacaagtgtgaggtgtcatttatcagaatgcatcacccca gtcttccctcctcaaatgattactgtaggctccactcaagagctcatcccagttcaagaccaccttcctcctccagagaagcaaatatat atatacacgtatatatatatatacacgtatatatatatatacacgtatatatatatatacacgtatatatatatatacacgtatatatat atatacacgtatatatatacacgtatatatatatatacacgtatatatatatacacgtatatatatatatacacgtatatatatatacac gtatatatatatatacacgtatatatatatacgtgtatatatatatatacattttttttttttgagacggagtctcgctctgttgcccagg ctggagtgcagtggcgcgatctcggctcactgcaagctccgccccccgggttcacgccattctccttcctcagcctccggagtagctggg actacaggtgcccgccacctcgcctggctaattttttgtatctttagtagagatggggtttcaccgtgttacctaggatggtctagatct cctgacctcgtgatccgcccgcctcggcctcccaaagtgctgggattacaggcgtgagccaccgcgcctggcagagaagcaaatatattga tggttgttaccaatacatgctcttgactaagaaaccttctttcttaattaatattgacaactttaagccgagtgcctgacatatattagg tactcagttactctttttcaactaaagttatgaatgatgattctaataaaagtaacttatttgtctactagttttattatgtttatttaa ttcattagaaaggccatggacatagtacaaaattcaaacaatataaatcatggaatgtgaaaagtaagtcacatgcccatcccagttctt catttccttacctcacaggtaacagcttttcctgtatctccccagagatattctatgtatattttgtttttaacaccaagctatatttaa aacaattatctttaataataatgttaatattgaaactggtaaagaaatatgtgtgtattatctcacctcaagcgtaaacaatagaacaag agagagcccattttgaaaattatggacaatgaatctagaaataatctcaaaagattttgcagtcaaaaaatagttcattagatacatgag aactgtcacttggtctcagtgtagagctattgcctcaactccctttattttcctaacaaaatcatcttgcttatcccatgaaatacgtgca tattgccaatcctacaatgccgcatcagaaccagaacccaactctggaacactaccttctcaagtatctttctgtctctttatggtaata tgttgaattaatattcacatctattatgactagtctttgatttgtagggttgctgaagtagtagcaccactgcagggctttctttagttt aaagaaagtaatcaggtgtccctactgtgtcatgatctccaccctcagctgggttctccagtctggttttaaagaacaaaacaaaaggct tctctgtctgagtcttactcaacccatcctctctactcataagaggtattccaaacctttacgattctcaaacttcctaaccgaccatct tattttcactctgcaaacaagctaacctcctcattcatagaaggaagtgcctcaacttcctccccgttctgaccttttctccctcccaaa tctatgtatctcttgtgacaaaatctataaccaccgctgtactttgagttctatttcttcattatttttgagggacctcaagtcctcaaaa aatatcctatcttgcctgtgtacttaacttttcttttattcttttctaactttcccttctcttccttggcacttgcccttccaggtatatg tgtgctcaggtctcctccaccttccatctgcctcacttcatggcatagggccttgaactatcacaaccaagctatgaaagagtagtcaac gcagtgtccccacttccttgccatcccattatcctagtttttcttttggctctctgaggagtccttcacaggctggttttcaggaataagt ctaaatgaatcactttcagttttcctaaacttctatgcctttgcacatcctcttacctctgcctagaatatctttctccttcttttccat ctttaaactctcacatcattcttcaagactgggatcagctctcagcatccggaagcctttgcctactagagacaaatgagaatgagtttg gtcaccttttcattttcttgtatcattctgtgctttattttgctcttctaagagcgttacatgcttcatttaatccctaaacaactgttt gaggcaagtacagttattatcctaatcatgcaaatgagaaaacagaggcccagacatgttgagtaactttgataaaagttaaa gaaccaataagtggaacagttgaggtttgaaccctggcagtctgactgtagagatactatgtttgacctactcccctctgcccccacccc atgtctgcccttagtttctgagcttgttgaatgaatgaacaggtggtagtctttttttgttataagactgatcagaattaagacaggtttaaa tttcacgtgtagaattttcaaaactgcaaaggcagtgcaaatctaaaaaaagaatggcattctcaggaaagaggaaaagtaagtgtgaga ataataataacaataaccaacaaactttagtaaatttagtaaatgtagtaaatttttacattaaaagcttttggacatacattatcatatttt atggccacatgaaatatattataatcccattttgcacataggaaatctgagactggcataaggagcacagagatccaggactttatatttt cattcttctaggattttgcacctcaggtcgatatgtatgagtaaactgggagtataatgggctctttaacagaaaaactaggaaagttttc ccactattattaattatttacataatatttttttaattttattattatttatactttaagttttagagtacatgtgcacaatgtgcaggttt gttacatatgtatacatgtgccatgttggtgtgctgcacccatcaactcatcatttagcattaggtatatctcctaatgctatccctcccc cctcccccctacataagatttataatggataatggacttcaatttctagagcaaaatggccccacccaaggatgccataatccttccagag ctctactgcaagatatgagatatacatatctaaaacttgttcttggtatttccaaagcagtcaacttttacacctgtttataatgcatcca aatgttgtttttatatggttgcatctcccatcttcttcaccaatagctatatatatttttcacaagagctgaaagagttcttgatgtaggaa tccatggtagagtttcagagaaatccctgaattcactgaaagttttatctagaaatacatgtgcaagtgaacacatcttttttaaaaaaaa tcattacctactttcttttttgagaagaaggtatttatttcaacagactcttgaaggagcctactcttcccactctcccacccccattaaga accactgtaggccgggcacgatggctcatgcctgtaatcccagcactttgggaggctaaggtgggtggatcacctgaggtcaggagttcgag acaagcctagccaacatagtgaaaccccgtctctactaataatacaaaaattagctgggtatggcagcatgtgcctgtaatcccagctact gcgggaggctgaggcaggaaattgctcgaacccgggaggcggaggttgcagtgaaccgagagagatcgtgcggtgccatttcactccagc ctgggcaacagagcgaaactccatctcaaaaaaacacacaaaacaaacaaacaaaaagaaagaaccattgtattagtgatggaaatgtgt tccctccctcccatcctggcaaccactttcttcctcctccatcataaaatatcttaaactaaactaaaataattttatttatcgatagt ttgaattttccctatcattgctacacagctaattgagaggtaccccgaggaaaatataaatggtacagtaatgcattgtagattttaataac atacttgacatcccaaattgttttcattggcttcattttaaaaactacatgttttaaaatcaagcagacactaaaagtacaagatatactg ggtctacaaggtttaagtcaaccagggattgaaatataacttttaaacagagctggattatccagtaggcagattaagcatgtgcttaagg acatcagcaaagtctgagcatccattttttaaaacgtagtacatgtttttgataagcttaaaaagtagtagtcacaggaaaaattagaact tttacctccttgcgcttgttatactctttagtgctgtttaacttttctttgtaagtgagggtggtggagggtgcccataatcttttcaggg agtaagttcttcttggtctttctttctttctttctttctttttttcttgagaccaagtttcgctcttgtctcccaggctggagtgcaatgg cgcgatctcggctcactgcaacctccgccttctcctgggttcaagcgattctcctacatcagcctccgagtagctgggattacaggcatgcg ccaccaagccccgctaattttgtattttttagtagagacagggtttcgccatgttggtcaggcttgtctcgaactcctggcctcaggtgat ccgcctgtctcggcctcccagaatgctgggattatagacgtgagccaccgcatccggactttccttttatgtaatagtgataattctatc caaagcatttttttttttttttttgagtcggagtctcattctgtcacccaggctggagggtggtggcgcgatctcggcttactgcaacct ctgcctcccgggttcaagcgattctcctgcctcagcctcctgagtagctggaattacacacgtgcgccaccatggccagctaatttttgta tttttagtagagacggggtgtcaccattttggccaagctggcctcgaactcctgacctcaggtgatctgcccgcctcggcttcccaaagt gctgggattacaggtgtgagccaccgcgtcctgctccaaagcattttctttctatgcctcaaaacaagattgcaagccagtcctcaaagcg gataattcaagagctaacaggtattagcttaggatgtgtggcactgttcttaaggcttatatgtattaatacatcatttaaactcacaaca acccctataaagcagggggcactcatattcccttccccctttataattacgaaaaatgcaaggtattttcagtaggaaagagaaatgtgag aagtgtgaaggagacaggacagtatttgaagctggtctttggatcactgtgcaactctgcttctagaacactgagcactttttctggtctag gaattatgactttgagaatggagtccgtccttccaatgactccctccccattttcctatctgcctacaggcagaattctcccccgtccgtat taaataaacctcatcttttcagagtctgctcttataccaggcaatgtacacgtctgagaaacccttgccccagacagccgttttacacgca ggaggggaaggggaggggaaggagagagcagtccgactctccaaaaggaatcctttgaactagggtttctgacttagtgaaccccgcgctcc tgaaaatcaagggttgagggggtagggggacactttctagtcgtacaggtgatttcgattctcggtggggctctcacaactaggaaagaat agttttgctttttcttatgattaaaagaagaagccatactttccctatgacaccaaacaccccgattcaatttggcagttaggaaggttgt atcgcggaggaaggaaacggggcgggggcggatttctttttaacagagtgaacgcactcaaacacgcctttgctggcaggcgggggagcg cggctgggagcagggaggccggagggcggtgtggggggcaggtggggaggagcccagtcctccttccttgccaacgctggctctggcgagg gctgcttccggctggtgcccccgggggagacccaacctggggcgacttcaggggtgccacattcgctaagtgctcggagttaatagcacct cctccgagcactcgctcacggcgtccccttgcctggaaagataccgcggtccctccagaggatttgagggacagggtcggagggggctctt ccgccagcaccggaggaagaaagaggaggggctggctggtcaccagagggtggggcggaccgcgtgcgctcggcggctgcggaga gggggagagcaggcagcgggcggcggggagcagcatggagccggcggcggggagcagcatggagccttcggctgactggct ggccacggccgcggcccggggtcgggtagaggaggtgcgggcgctgctggaggcgggggcgctgcccaacgcaccgaatagt tacggtcggaggccgatccaggtgggtagagggtctgcagcgggagcaggggatggcgggcgactctggaggacgaagtttgca ggggaattggaatcaggtagcgcttcgattctccggaaaaaggggaggcttcctggggagttttcagaaggggtttgtaatcacagac ctcctcctggcgacgccctgggggcttgggaagccaaggaagaggaatgaggagccacgcgcgtacagatctctcgaatgctgag aagatctgaaggggggaacatatttgtattagatggaagtatgctctttatcagatacaaaatttacgaacgtttgggataaaaagggagt cttaaagaaatgtaagatgtgctgggactacttagcctccaattcacagatacctggatggagcttatctttcttactaggagggattatca gtggaaatctgtggtgtatgttggaataaatatcgaatataaattttgatcgaaattattcagaagcggccgggcgcggtgcctcacgcc ttgtaatcccttcactttgggagatcaaggcggggggaatcacctgaggtcgggagttcgagaccagcctggccaacaggtgaaacc tcgcctctactaaaaatacaaaaagtagccgggggtggtggcaggcgcctgtaatcccagctactcgggaggttgaggcaggagaa tcgcttgaacccgggaggctgaggttgtagtgaacagcgagatggagccacttcactccagcctgggtgacagagtgagactttgtc gaaagaaagaaagagagaaagagagagagaaaaattattcagaagcaactacatattgtgtttatttttaactgagtagggcaaataaa tatatgtttgctgtaggaacttaggaaataatgagccacattcatgtgatcattccagaggtaatatgtagttaccattttgggaatatctgct aacatttttgctcttttactatctttagcttacttgatatagtttatttgtgataagagttttcaattcctcatttttgaacagaggtgtttc tcctctccctactcctgttttgtgagggagttaggggaggatttaaaagtaattaatacatgggtaacttagcatctctaaaattttgccaac agcttgaacccgggagtttggctttgtagtcctacaatatcttagaagagaccttatttgtttaaaaacaaaaaggaaaaagaaaagtggat agttttgacaatttttaatggagacgggagaagaacatgtagaaaaggggaaatgatgttggcttagaatcctaactacattggtgtttaa tataggaacatttatttatataacattttaaagtactaaattcatattagtatattatcaaatggatatattatcaaatgggtttaagcatcc tacacattttaattcaattgattcattttctttttgctttggatttctatcatgatttaaatatttacatatgggttactttttagatttttc atactatgaaatataagaaaaacctttaaggctagttttatgaccaagacgaaggacttcattgaatacacaaaacaataaatatactgca acattttgtctttctttttgtagctgcaatttggtttgcttatactttctctttgtctctttgaaaactgagtcagtttcactttctcagga caggatttaataaccataatataatttagtataattccttgatttaggcaaattatgcaatttgtgtttagtatgaaatgtacctaaaaata agtaactcctctttaacaccaccatcctcaaactaatataacaaataacagttatcctaaaataaattgtctacttccaccatgcagcact caaattttaaggttgctatgactgcagacagtattttaaaattcctctctggaaatggctttgtttccaagatgatttaggaaccaaagag gtgaccatctcttgtttaatgaactctcaaatcataaacctgggaagtgttttagtttcctactgctgctgttacaaattatcacaaatgt gttagctaaaacaaacacaaaattattattttacagttctagagatcagaagtcaaaaatgggtccacaaggtttcattccttttggaaac tctaaggggcaatctgtttccttgtcttttccagcttctagtgaccatcaaattccttggctcatggtctctgtattttctctgtggcct gtgcttccattcttgtatcttctctctgactgtgaccctctaataaaaacacttggggttatgttgggcccaccctgaaaattctggataat ctccctcaagaccattaattaaatcacatctgcaaagcctcttttgccacataagttaatgtattaaaagtttttgaggattaggacatag acattgggggtgggggggcattattcagcctaccacaggaaggaattttagggttaattaaactagccttcttattttatacttgaagaa attgaagttttggaattggagagcattatgctaaatgaaataagccaaacacagaaagacaaatatcacatgttctcacttatctgtgaaat ataaaacaattacattcttagcagtaaagagtagaatggtggttactagagctggggggtgggaggaatggggagatggtaatcaagatata aagcctcagttaagatgggaggaataagtttgattgttttttttgagatgtgtttcatagcatgatgaatatagctaaatagtaaatccc aaatgctctcatttgacaaaaatgtcaaatatttgagatgatggataggttacttagcttgacttaataattccccattgtgttcaaaga tcataacttcatattgtaccacataaatatatacaactgtactatcccaatatataattttaaaactaatataatgaaaaagaaattgaa gttcaacattcccagaagctaagtgtaacttaaaagttttgtgagaatttgttttaacaaacaaacaagttttctctttttaacaattac cacattctgcgcttggatatacagcagtgaacaaaaaaaaaaaaaaaaatctccaggcctaacataatttcaggaagaaatttcagtagt tgtatctcaggggaaatacaggaagttagcctggagtaaaagtcagtctgtccctgcccctttgctattttgcccgtgcctcacagtgctc tctgcctgtgacgacagctccgcagaagttcggaggatataatggaattcattgtgtactgaagaatggatagagaactcaagaaggaaa ttggaaactggaagcaaatgtaggggtaattagacacctggggcttgtgtgggggtctgcttggcggtgagggggctctacacaagcttc ctttccgtcatgccggcccccaccctggctctgaccattctgttctctctggcaggtcatgatgatgggcagcgcccgagtggcggagct gctgctgctccacggcgcggagcccaactgcgccgaccccgccactctcacccgacccgtgcacgacgctgcccgggagggcttcctgga cacgctggtggtgctgcaccgggccggggcgcggctggacgtgcgcgatgcctggggccgtctgcccgtggacctggctgaggagctggg ccatcgcgatgtcgcacggtacctgcgcgcggctgcggggggcaccagaggcagtaaccatgcccgcatagatgccgcggaaggtccctc aggtgaggactgatgatctgagaatttgtaccctgagagcttccaaagctcagagcattcattttccagcacagaaagttcagcccggga gaccagtctccggtcttgcctcagctcacgcgccaatcggtgggacggcctgagtctccctatcgccctgccccgccagggcggcaaatg ggaaataatcccgaaatggacttgcgcacgtgaaagcccattttgtacattatacttcccaaagcataccaccacccaaacacctaccct ctgctagttcaaggcctagactgcggagcaatgaagactcaagaggctagaggtctagtgccccctcttcctccaaactagggccagtt gcatccacttaccaggtctgtttcctcatttgcataccaagctggctggaccaacctcaggatttccaaacccaattgtgcgtggcatca tctggagatctctcgatctcggctcttctgcacaactcaactaatctgaccctcctcagctaatctgaccctccgctttatgcggtagagt tttccagagctgccccagggggttctggggacatcaggaccaagacttcgctgaccctggcagtctgtgcaccggagttggctcctttcc ctcttaaacttgtgcaagagatcgctgagcgatgaaggtagaattatggtcctccttgcccttgcctttcctttttgtgatctcaaagcat cctccctccgcccccattccatggccccagttccctactcccacagctgtctgctgaaactgccaacattactcaattgtttctgggggga ggaacatttttttttgaaacaaaatagatatatgaaacagtacacgggaattaacacgaatatttaaggtaaaacatgaccttgaagatt atgaaatccatcttattttggcccagaacgggggcattgggctccttgggccataggggagctggggaggacagggtgaagagttagctct aagccctctgcttggagatgctgtaaatacagaacgcaaaatcaccttcgaagttaaagacgcgaagttcttctttactcggcccctcct cccctcccccccgccaattccctccagttacagctagcatccaggtcccgggaggtgaagaaggagacttcggctccagttacagctagca tccgggtcccgatttagaaggagctgccaattacagcgcggttccagggctgagcaaaaagcctgaggagccaagtgggagagggagtaaa actactgaattgggccacaagcaaatgaataaactgaacgactcttaaccaaacctaatatatttaatccaaacacacaagtctttcatt tcttccctcctcccttccttctcttactccccaacaccccctcttcaagcacaattaattatatggttagattctactgcgtgatcagcc ctgttctaggtggtgggcacgccaaggtgaatgagaccaaacaagagtcttgccctcatggggtttacatttggagacagagtcgatctgt gtgcccaacctggagtgcagtgcgcgatcacagctcactgcagcctcaaactccctggctcaaggggttctcccacctgagcctcccgact agctgggaccacaggtgcacgccacgacgcctgggtttgtttgtttgtttaatagagacgaaggtctcaccatgttatctgggctcaagcga tcatcccccctcctcctcctaaagtactgggattacagtcccaagctatcttgcccgacctgggaaacagacgttaaggaagataacaatc tattttcagagagcgagtttataaaaccaatgcaatgggtaaatatgaagtgtgaataggaggagaagctaaagagtggtcggagaatcta atgcaagctacgggagaaagaaactcaagtgcaaatgctgcctcaggaataaacgtaaaaagagactttcaagtgcaaatgctccctcagg aataaaataatcttgagactctcaagtgtaaatgctgcctcgggagaaccgaacggcgagctggagcccatacgcaacgagattagagagga aggcagaagccagagcacatgaataaatgagcatccattttgtttcagaaatgatcggaaaccatttgtgggtttgtagaagcaggcat gcgtagggaagctacgggattccgccgaggagcgccagagcctgaggcgccctttggttatcgcaagctggctggctcactccgca ccaggtgcaaaagatgcctggggatgcgggaagggaaaggccacatcttcacgccttcgcgcctggcattgtgagcaaccactgag actcattatataacactcgttttcttcttgcaaccctgcgggccgcgcggtcgcgctttctctgccctccgccgggtggacctggagcgc ttgagcggtcggcgcgcctggagcagccaggcgggcagtggactagctgctggaccagggaggtgtgggagagcggtggcggc gggtacatgcacgtgaagccattgcgagaactttatccataagtatttcaatgccggtagggacggcaagagaggagggcgggatgt gccacacatctttgacctcaggtttctaacgcctgttttctttctgccctctgcagacatccccgattgaaagaaccagagaggctctgag aaacctcgggaaacttagatcatcagtcaccgaaggtcctacagggccacaactgcccccgccacaacccaccccgctttcgtagttt tcatttagaaaatagagcttttaaaaatgtcctgccttttaacgtagatatatgccttcccccactaccgtaaatgtccatttatatcatt ttttatatattcttataaaaatgtaaaaaagaaaaacaccgcttctgccttttcactgtgttggagttttctggagtgagcactcacgccc taagcgcacattcatgtgggcatttcttgcgagcctcgcagcctccggaagctgtcgacttcatgacaagcattttgtgaactagggaagc tcaggggggttactggcttctcttgagtcacactgctagcaaatggcagaaccaaagctcaaataaaaataaaataattttcattcattc actcatttattgtcaacatttattgagcacctattacaacaatttcatcgcatggaagacagcatcgtttctgacactgttgtttcatgta tctcttagaaaaacgctgctattagacatctaacactatttatcttgaggtgataaaatatcaaaagccgtgtctcaagatcgatgaaatgc ggttaaaatgatgaatagaaactctagggggacctcatatcgatagactcgagactggcacatctggagatccgtatttatccggcttccc cttccagatcacgcgaggtttgggatattttgctcaccaggcctcagccaggtaactgaatccagccaaccctggcccatagtctcggaatc cgactcggctcccagtccccgcctcggcgttctgagacccccaggctggggttccagagggctgtgaggttgcgaatgactgctgccaaac cggaaggaactctgcggttctctgccacagtgggattgttgcaggcacgcggctcagacttcactgaggttgggagatgctcctgtcc acgctgcctcatcccgtgctggagcactgcacctctatttttttttttagggtacacgc Protein sequence: SEQ ID MEPAAGSSMEPSADWLATAAARGRVEEVRALLEAGALPNAPNSYGRRPIQVMMM NO: 348 GSARVAELLLLHGAEPNCADPATLTRPVHDAAREGFLDTLVVLHRAGARLDVRDA WGRLPVDLAEELGHRDVARYLRAAAGGTRGSNHARIDAAEGPSEMIGNHLWVCRS RHA NOTCH1 SEQ ID cctcgcggtcaagtgctcgggcaatcacggccagggatgtctggcggcgatcgatcctctggacgcctaaagccgcggccacggg NO: 309 gccctcgggagggagtgaagccgctgggctaggggcgcacacacggctaggccactctcccagagccctgccccgggcccggg gtcccccaacgtggcctcagctgctccccgcccggcccagcgcacggtgcacacggctgtccgcggcctcgccctccccattccgc cccgggctcctccgcttattcacatgcaaatttcagtcgccagttgtcgccgagcgcggcaaccgccagagccggatccttccgcag ccccggctcaaacttttggcctctgaaaactttcaaacgagaagtagtcccaggcgcccgctcccgacccacgccgcgccgccggg tccctcctccccggagaggctgggctcgggacgcgcggctcagctcggggaggcgcaaaggcggacggggcgtgcgggagga ggtggccgcggagggggcggggggaccggcgggggtggggccgggcggggcggggccggcgggggcggagcgcacctc gactctgagcctcactagtgcctcggccgcgggagggagcgcaagggcgcggggcgcggggcgcgggcgcgggcgcgagcg cagcgaaggaacgagccgggcgcggagccgggcccgggggcctgcgagagcacagcgccgccagccagccggggaagaga gggcgggaccgtccgccgccgccccgggaccgtacgccgcgcgtgtgcgtcccagccccgccggccagcgcaggaggccgcc gcccgggcgcagagggcagccggtggggaggcatgccgccgctcctggcgcccctgctctgcctggcgctgctgcccgcgctcg ccgcacgaggtaggcgcccacccacccgcgagcccccactttccgcgccctttggaaactttggcggcgcccggcgcgcgcgcc ccacggctgggagcgggcggcggggaggccagcatggagagggaaaagcgggcggcccggggcgtggggttctggagtccc gggatcagggaggaccgaccttccccctcgatccccccgtggaggcggactcgcgccgcccgtgcctggagccgagttaggagg ccggtgtggggtgctggggccccggaggccctactccgggcccgcccttcacccgccgcgcgtggggcttgccgccggtcggcc gggcgggcgggctgcctactatttttcgatttgaatagagtcggttttggtttcctgttgcttctccgggccatttatcttctttcttctt cgcctctggcccacgccggggcggatgttggggcgcggagtgtgggctctgcggcgccgcgttcgccttcactgacccgcgcggccggg ctgggtccccgggctcccggtcgccccgcccgccggtgccccccagcccggctctcagtttgggggaggggttgcgtaagaagcc gccgcgcccggggggactgaactttccttttgctttgcggagttgaagtttggaaagcttgggggcggagagcgggacgcgggtgg ggggctcttacatttctccccgccgcacagcgagcggggtctctggggaatcgagtgattaatccactctttctccgagagttggaggc gagaattatctgtcctcttccagaaagtgcggctctgtgtcacacccccctcccccgtttctcagccccggtaagatggggagggagg ggcttgagtaattgatcccttctcgagatggggtcgaattccttccgaatgggggaccttcatccccctcctgtgggtgtatgggggctg ccctggagatgcgcgcccgcggaggcaggtattgggtgtcggcggaggcggggccgcgtccccagggtgctgtcccggtgcccc tggaggcggccccgactccacaatgggccgctctgattctgaggcggaggccggcgctttgttggcgggcggggtagcgggcga gcagctgtcgcattttcccacgggcgggagctgagtgtggccaccccccctccccccgtccagctctgcccccttgcagaacgggttt gaacagaggcaatctcggcggctggatggggggccctgccctgccagactcctcagtgagcctccagggtggggggcaacgcttt ggagagtagcccactttgtctctgcttttcccccactgcgtccaggcggcacatcaggccaccccaccctgcttcaagcaggacgtgtt ccctgctgctccccttccccccatttctgactacagaactctgggcagaatgttgacgcccctttggtttcatggaggggcttcctgcgg acccgtgccccagcaacccatgatactgagcagagtgcgtccggggtagggggcggacgatgccccttggctgggtgcaggtccc cgcccccagggtaaccagggccttcggtggtggtggtgcgggtgagactgacctctcttctcctgcccctgcctaggcccgcgatgc tcccagcccggtgagacctgcctgaatggcgggaagtgtgaagcggccaatggcacggaggcctgcgtgtgagtaccacccctgc gggacctgttgctttgtcgagggcagagcccctgccttctgcagccgcgcaggggacagaacactaggccattgtcttctggagatg gcccagagtctgggcacggtcacgtgctgacttttattggacaaagtctggcaattacttaatcaccagcaattatgctgcgtgtggagc tggtctggctgctgagggcttgggcactcctccgtgggccacctcaggctccggggtcactaagtgggagtcccagtaccccaattct tctttaagtccccaagaaacagttgatctgccaggggaagctctggaccatttactgagaggcccagcccccaccaccctgccaactt ggggcgcctcctctgggcgggtgatgcctcctgggcacaggtgatgcctcctctcgggggtgatgcctcctaggtgggtggtgatgc ctcttgggctggtagtgatgcctcctctggcacaggtgatgccacctcctgggcgggtgatgcctcctctgggtgcgtgatgcctcctg ggctggcggcaatgcctcctctgggcacaggtgatgccacctcctgggcgggtgatgcctcctctgggtgcgtgatgcctcctctgg gcacgggtgatgccacctcctgggtgggtgatgcctcctctgggtggtgatacctcctctaggtgggtgatgcctcctctgggtggtga tacctcctctaggcgggtgatgcctcctctaggcacaggtgatacttcctgggcaggtgatgccacctccgggtgggtgatgcctcttc aaaggaaattgactcttcaaaatcggacccgcctttgggctctctgtttgccccagctccctcttcccggcctcaggctggggctgggg aagagaaggtggaactcatgttttggcgtggggtgtggggtaattattgagtctggtttctgtcctggcatctgcaaactctgacctcaaa atcccgtggcccttctggtctgccagcctcagatgtaaaatgggcactgagggggccgggaagtcgtgcggaaccgctccctcctgc ctgcctgtgagtgcccatcagccgtggggaactgttctgctgtggccgatgcccttcaacttgagtccatccctgatgagaataatgag gaggtgtttggtgtctggaaaaagagttccctccgccccccaggagctacagatgaagaaatggctccccagccagtctgcgcgccc tcattccaactgcagaaggtcccattcccttgacaaccccagcccggctcctgcctgtgccgactgcccagcctgtgggcagcaggc cccctccctccccggcccccaccctccccaaactgaagccaattaagcaggtctgtgagcagtttaatggacaaagccgattgtgtctt tgtcagacactaatgaatgcgcactacttttttttttttttttttttttgctttttggggcccccttctccctctgcagaagccgcacggac gcttgatgccctaaatcttgtttcctttcattcagagaccgacagctgggatccaggggaagtgcagcagctattgttgggggaggcggattaa tgccgtgtgattaattatgcgtggcttcccagaatgtataacccccttccccgccactgcgctcccggctcggactttgcctctcacccat tcggggctctgggagaagcttcttgctcctgcagaggactttgggagggtgggaggaacaccgtgctgggaaaattgggggcctgtt gcttcttctcagtggcaccagggagtaggcacagcctggcccacaggggtctgggactgaggtctccctgggccagagcgctgtgc tttggggtacagattgctgcagctccctggtgtctgggttaaaaccctgccacagggccgcaaaacactggtgtcatgggcccactttc ccattccccaaacctgggatcccggtgagctgtttccagcccaaggcaggccgccgaccatggctttggtgcctggccttgtccccttt gctggggcctggcgccccctccttcttggcgggaagcctgcttcagtttggcctgacaggcagccgagctcagcccacagagaccc cattgtgcacaccccacaagtccaacgagaagatgggacggaagcgcgtcctcaggctcctgcttctgggccgcagagttggaggc cagggccacagccgagcccggcttgtggaggcacaggtcagggctcacacagcagtctgggggacattgcagccgcttcggaga aggccccgggctccctccctgggcttgagccccacccaacatacacccctcaggtgcctctgccttcccgttcctgcccatccttctca gaaggcccaggaggtctgtccaggagttagccagggcacctgcaggaggcctggctcccccagcaaggggagaaacccacactg tttctcgaaagagtctggggtgcaggctcttggagccctgccagcctgagtgggaatcctggccccgtggtatccagctgtgaccccg gaaagagctttggcttttggtgccatttgctgcctccgctgtagaaggggcatggtcatgatgggcgtcacagtgcctggctcactagta ctgggtgaatcatttctttccatcccaaagagaaggaaccttcaccggaagctgccaatcaggtggttgcagggcccgggattggaaa aagtggttttgttggtgcaaaatgccttcccaggaggtcagagtgtcgccgtggcctggtcagccagtggccaggtagaaagcacctg gcagcacatgctggtgggcggaggatggttggtctctgggcagctgctgtgcgaggtgtggccgtggtggctgtggtcggagatgg tggccttctgagcaggcccagggggtcactgtggcccttggctcttctctagccccctcatcagctccgtcagtgtgagcaaggggta ctgaagagggctccaccttttttctctttgttttgaggagagctgggaagagctgcagggaccctatctgtctgggtgagactacgtcctt ggtctgaaacgctctcccagggctgcctgcgtctgggctgggcgttgaggcagggggcagctaagatgttggctgctgacggcaga gtcagaatggcacggggcgtcttccaggagctgggagaaatgggggtaggctgtcacacccagcacctccaggaggatgtccagc tgcctgctgggtggtgcgctttagctttggacaacctcgtgtctgaggcctggcctggccactagctctgtgaccttgggtcatcaggtc atgcaagcctcagtttccccagcatgtcagccgggctgatacacgctggatctcttggtggtgtgtgaggattgcatgagaaccgtgtg gagagggcacctggtcagcgctcctttcttccccgccttctccccgttggctgacagtgctggtttccagagcctccttcctgaatgttta attgattgacgagtgaattcagagaatcctaaaggtgctgcctggtgggctgggcctggcactgcctggggagggacttgccggctct ggggaagtccatctcccaggatggctccagctccggggaagtccatctcccagggtggctccagttcacagacacgtttgagcgcct gctgtgtgctgcgttctgctccaggccaggcccccggtggggacagggcagacatggcccctgctcctgtggagttcacttcccagc aaggcgtgagagaggcacctagctcacatgtgggacatgttgggggtgtgccggcccggtggtggcctggcatagctcccggcag ccccgtaaagctgtctggatcaggcaccgataagtgagaagaagagacccagagaagtcgccatcagccccagggtcacacagca gtggcagaattcctactagccctgcccctctccttctcccaagcgaatgtccctaaacacagccccagccagcctgagctgccccgtc atttcccgactacaagcggactgggggcgtggcttccccttaaaagaagaggaaggaggctcaggcgggaagtgacttggccctgc agccggcctgggaggctggggagggacggggtagctcctgtcacccggtctggctctttccattgagtcacctgcctcgtcttgggc gtggccaggggaggaacaggttgattctcctcctcatgctgagctgagcggaaaggcctgtgacaggacctcctgtttatgcagaac ctggtcttcaagggcgccagggttagaaaaacatgtttaaaaacatgcagcatccgaggtgggcagatcacctgaggtcaggagttc aagaccagcctggccaacatggcgaaacccccgtctctactaaaaatacaaaaattacccaggcgcagtggcgtgtgcctgtaatcc cagctacttgggaagctgaggcaggaggatcctttgaacccaggagacggaggctgcagtgagctgagatcgtgccactgcactcc agcctgggtgacaaagtgagactctgtctcaaaaaaacaaaaaacaacaaaaaaaaccccacacagtgtcaaatagaaagtctcacc tactcagactggggagacatcaggaacaggaaggcctggggcggagactggggtgggagcctggagtgctctgaaggaagctgg ctccttggggcaggccctgcctctgaacagcagccaacatgttttccattgaaaagaataataatagaaaaagagagagggagaaag aaaacagcaaccctggcaggaagagggtagggcagggcagacagtgcaccaggggaagtgcttccacagggcagagctgggtg cggggacccccagatgcgggcggcaaactggagaccccacaggagccgggactggcttcattcctcattccctgcacctgtgatgg gcagcagcggccaccggtgcccactgggcagttccaggccgggccgagggactgcacccttttgctgtggggaggggacctttcc tgtttcatgtctcgtctataaatagtgaaaggatgtggctttgcagaatcgattgggcgtctagcttgccttcctcaataatgcagcgatga cagctccacactcaggacgccgctgttacccagcctcgtaaaagccctggcagacggccgcccaccctccaggtgtgccagtgcag ttttcatccccagcgcacaggtgggaaagctgtggctgcgagggcgtggggatctgaggcaggctggcgtcagctttcaccttcatgt gcaggcatatccagaactttccggatctttccccgggctgaggaggaaataccagcagctttaacgagcggctggctttggggcatca cacctgtgggccgggctgcgtttgacggcctcgagtttcccaggcagcgctggcgctttctcctgtggggcccggagctccgaggg gctgtgaagcagggctggctctgatttgctgtgctccccacactgtcccctccctgggcctcagtttccccatctgtatgtgattccccttc caactacagccagcgggaccctggggcgggctgaaggcgtggtggtggctgtgggccctgcggcgaggcctgcgctgggctcg gtggtcgccccagctggggaaggggcagtggctgcaggggtgggagggtggagggaagcccttttccaaagttgcctggttgggtt ctccttgtggccctgccccaccccaccctgctccctgggagcaaagggagctaaggactggtttggggatggatatgtatatggagg gattctgggtgatgacttattcatattacaaaatttgcctttttttgtttgttttttgagatggagtcttgctttgtcacccagactggatta gagtgacgcagtcttggctcactgtaacctctgcctcccagattcacgcgatgctcctgagtagctgggactacaggcacataccaccactcc cagctaatttttgtattcttagtaggggcagggtttcaccatattggtcaggctggtctcaaactcctaacctcaggtgatccacctgcctt ggcctctcaaagtgctgggattacaggcgtgagccactgtgcatggccacttttttgttttttgagatggggtctcaccctgtcacacagg ctggagtgaagtggtgggatcacggctgactgcagcctcaaactcctgggctcaagcgatcctcctgcctccgcctccagagtagct gggaccacaggtgtgcaccaccatgcctagctaattttaaatttttttgcagaagcggggtcttgctatgttgtccaggctgaaaatgtga cttttgaactaaagcatgctgttgcttgacatctagtccattcacaagttcattcaaccactatctagttccgggactttctcactgcacaaa acagaaactgtacccaaatactgtcactctgccctgactgccccagcccctggcaaccactaacccgctttctgcctctgaattcgcctc ttctggacgttgcctatgaatggaattgtgccataggtggtcttttgcccctggcttctttcactgagcgtgatgtgtttaaggttcatccatg tcacgcgtggactagaactgcattcttctgaggactacattttagcctccagaagcatcagggactggccagaggtggaccccagcat cccagctcccagacagtccagaggaggcaggtgttgggagggcgtccggggtggcttcctggaagagatggcgctcactctgggc cggtgcagccacccgacagccacagagccccctagagggagtcctggcatattcccaggtgaacctgtaggatggactggcgtgc ccgcggcccctgtcctgctcaggactccgagacaggctcacaacctgtccctgggcctcactcatgccagccgccaagctgttcagc atcccttagcccccaggaggcacacagtggggccgcggtcactgagcgtccacacgcctggtgctgtaagaatgcaaagtagagg gtctttattgagcatctactacatgctgaaccctggggtatgggtgggcacggcagatgcaggcccccctgggcaggggaggagcat gccccttaagaaccaggccacaggatggacgggcttggccacaagctaggggtagtcactgcagccttcatggcctcctgtgtgcaa agtcagtgctgtgggaccccatctgggtgcacctgctgtggctttcaagtgaggcccaccacggagcagcttccacgctggctggga cagggtcagcggcagggagctccagaccaaaggagcggcggtggctggggcagcggggggcagtgaggctttggacacatcca tggggcgggcaggcatctcagcccccacccgcccagtctgcctgctccggggccttcccttccctggacagagctctgtgtgattgg ctccaggctgcttctccccctggaccctcagggtcagcttagagtgactggctatgccctggcctagatccaggcccgtgtgacagga gagagctaggaacctggcccctcactctgagacacagatggtgtcctctccaagttgcccttgggacggggaagcgccatgtcccct gcagggcaccaggccagccagtggcccttgacagcgaggccctgtctgcaggcagctgggggacttctccctgtcgggaggatgc gtgcgtgttagcatcggggtgcctggttgccccaggggtggtgcagggcggttataggcggtcctagcattttggggttgctttcagtg tatctccgagacctgtggaattctgggaggagaaactccccagcagtgtcctcagtgcgcccccctaccctgtacccgttgctcctgg gagggatcctgagtgctcccccccacccaccatgcacccattgccccaggagggatcctgagtgctcccctctccacccaccctgca ctcattgccccgggagggatcctgagtgctcccccctccacccactcccaccctgcacccattgcccctgggagggatcctgagtgat cccccctccacccaccctgcacccattgcccctgggagggatcctgagtgctcccctctccacccaccctgcactcattgccccggg agggatcctgagtgctcccccctccacccactcccaccctgcacccattgcccctgggagggatcctgagtgctccccccgacccac cctgcacccattgccccaggagggatcctgagtgctcccctctccacccaccctgcacccattgcccctgggagggatcctgagtgct cccccctccacccaccatgcacccattgccccgggagggatcctgagtgctcccccctccacccactcccaccctgcacccattgcc cctgggagggatcctgagtgctccccgcctaccctgcacccattgcccctgggagggagacttctgtagaggcctcacctctgaacc ccccactcacaggctcaggggcccagggctgggggcgtcctgccgtctacactccagattgagcccgcacggttaaacagggccc tctccgcacgccctgcctaatgcaaagcggtggcttcgggtgggtccccaaggggccttgtgaaccctgggggcctggcccctccc acctggctgcacccgctgggcgggagatcccatcctccgagtcctcagcccctcagcctcagctgcagcccagacaccagggctct gtggggcgggggtggggcagcggttccctcaggtctgtggggctgccctgctgtggttcctgcaatttcctggaaggaggtctgggg cctggtgtccagtggggcctgtggggacggactcgggggctctgtggggagggactgggggctctgtggggacggacttggggg ctctgtggggagggactggggggctctgtggggagggactgggggctctgtggggagggactcgggggctctgtgaggagggac tggggggctctgtggggagggactgggggctctgtggggagggactgggggctctgtgagagggaactggggggctctgatgtg ctgggcgtgcacaggggaggctttgcctctgacatccacacctgcagttcccaggcacacacagcggccccatcggggtcctcgca atcacagtctgtgagtggccactaccaagggcgtctccggatcaggccaggcccagatgctgcccggacccaacccccacagcgc ctgccgccacagtagggaccaatggccacagaacactcacccggcctcacttgcacggggctgttcagtctggtgggcaccaggtg ggcgctgttctcgcccagtgggaggcctgccagctccttggttgctagttgagatttttccccaagaaatagtcgtgttgttccttctgctc cgcctggcactgaggttggtgacacgccccgaccttgctctaattggcagatgagaatttgtcatcagatgtcgaccctgtctacgcag tcccctggcctcggagtccattgtgcatctctgaagggcttgaattggttgtttaaagctgggtaaatgcccccttgacattctgttgacac tgtcaatttgctgaacaaactcttccacaggtagcacaggaggccagtctcgccccagcctgagccaaggcccagggaggcaggtc ctgtgtggagtcagcctttttgtgagtgcagcggccctgcaggatgcaccgggtttgagaagtggactcccccgtctcccgccacatc ccaaaagccctcagccatgagggcaggacgaagccgacggtcgccctcgtgccgagatgacaaccgggcacagaggcggtggc tcccagcttcctgccctcttccccgtagtggggttacccagcgtgactcatggcccgagccacaggcacccgcccagggagccacg gcggggagacctggctttattagagatgcgttcgtgcctcatcaagtccaaaggaggaaactggcgcgtccctcactttccctgaaac aggctcccccacagcccaagcggtcaggttaaggtgcatttgcagacagatgccctagtaggaggctggagcttttggcccagaattt tccctgctgtaggcccatggtcaccgagcctgcccgggggctgaggccctgaggaggtgccattgccccacccaagcctcagaca agtgtcctgccctcgaccctgcgggaggcagcagctcaggccctgaccctggccagagagggcagctctctgaggctgctgtggc cgtggcaggcaggggctgggcttgccgggccgtcgagtgggcacagagactatctgggaaggaggtgggcggtgagagctgact ccccgcagccgggggacagatgagccccgtgccgcacagcagggccgcgtggtggggccccccaagttccgctcggtttcagct gttgacgagcggaaacacacagttactggaaatgagaggtttgggggagccgccaggggttgaacatgtgtggtttccctgacggct tttcagtaagagatgcttgagctcaggctgggcactggctggtgcgcggagggggctgggccagagtcccccccttgggcctccact ctccatccttatagagggacctgggttggtcccagctcccggggtgcagcccctgggtggcccctcctacttccccttcagtggtcaga aggaggctggcctcattcctgtctgacggagagccaggtggggaggccgggcggggctgagtgtccagggtccacatggtggcg gggctcaggccgtggcgaggtcctgtcctgggcctccagggtctggcccagcacagagctctgcagctctctggtggacgggcctg gctgggctgtcggcagccctggggccaggtgggcagtgggaatggaggtggaggcaggtgggtggctgcaggtgactggaagg cttggctccagcctgcaggggctgcagtccaggccggccacccccacgagaccttcccccttccaggtgtggggtggcactgggtc aacaacacaggccacggacttctttcccccacgtgggccgtgcgaccccgggccgccaccacctctctgagctcccctcctccttag gagatgggaagcggctggggccatgaagtgtcccccagggctcctgacagccccagctgccgtcctcgtggctgggcgactgccg cgaattgttgcgccttcttttgagttggctacggagtcatggcggctgtgggaggtgtagctgggtctcgggcggcacgcccggctcc cccggggatgtgtcatctgtgtcatccaccctggagccccttcctggtgctgtgggggcccctccaggaaggtctctcccagaggcca gccacaggctctggggagactgtgtgggatttgcatgcagttgtgtgctatttgatcgaggtcatgtgtgggatttgggcgaggtcctgc gggattgggtgaggtagtgcgagatctttgggcgaggccgcgaggcactgggcgaggctgggcattattcagtgccttggaggggc agaagttttaggacctgccaagtccagcccaagaatccccaactttccccacagggaaaggtaccccagacagtccccagcctgtgc cagctgtcttgggacaccctggtgtcccccaggttgtggcactgcaggcccctcccccaggtgcccctccttatgggcccctcccagg gctgcaggcacctgcctggatcccacttcccaagtgtggggtaatcccaggcgcccggtgatcacgctgggcccgggcagagtga ggctctgagggggtgccaggggcacagtgggccagtatgtgagccgggctgggcgctgggactgggccggcaggcgagcggtg ttccgagaggaggattcctgggctcggaggcccctctgtgcctcgtgtggtcagagagcagggagccggcgggtgtgatggggat gaggtgacctctggggccaccactgaagccgccgtccatctgcggccgaatcgggaggcacacagaggtgtcctggtggtttcccg ggacaggcgggaggaggcgcaggagggcggcgtctgcccccgggatgccaggagtgctcctccggcagcgtgggcttcggctt cgtccctctttcaccagtgccgacgccccgcgggtgctgtgggtgaaggggcatcggtgccctcctgctgggcgggaggaaggaa ggggagcgggaggctgggtctctctcgcccagggctctgcccccaccaccctagggttttgtgtttcggtggagctgtaagaatgcttt gtctgctccagacttcccggtcctgggagatcaggtggtcaggcaaaacatccagaccgtctgggtggggctgggggtgcggggg acacgcgccttcctcctctttcccagggagcttctgccactcctgtgccaccctggggagctgccttgccctgccccgctctggtcccc atcagcaggtgtggttctgagctgccttcagatccgggtggtgtgctgggcatagctgccccgttttctcacctgtgaaatgggcccatt acacgcccccagggcggttgtgggggtcccatgagacagagcttggaaagcccgagtgtaggtcaggccaggagggttgtgggct cagtttcttcactgctgggctggtgtgtgccggacagatgtctcgggatcccttccctccctgcagcattcttaacaaggacgctggaca tggagtcagcacctccgggcgactgccctgctggctgtggggctcactccagaggagaacaggagcccctccggggagccccttc ctgccacccccagcgacaggatctgttcaggagcagtggtgggaggttgccagaagcatgtctggcgctggcccggagcgcagcc tgtgatgcccgacttcattcatggagtgtggtttgagccctccagcagcgctgctgtgggcctgtccattgccttccctgcctctgagcc atcgtggggtgcagggctgggctgggtcttcttgggagcagagccccggcgtgtcatgaccatcttggcctctccaactcaagaggtt tgtccacatgggtccctaggggccgttggggtgacccagggcaggagacacatgcctgcttttgggggatccgcgtgggaaattccc gcagtggagcagcaggtggggctccagcaggcgtttttcacactccaaacagcctgcgggggcactttggtgacacctagttcctgg ggtctcagagcctgctgggctgtgtcttggcattttggggaggggctggccacatgcccccagagtggccaccccagctccgccatc tccacgggccttctgtcggtcagatgcagacagccgttcccatgtcgggtgagggcattgttttcccggctgcgggaaaggtagcttc ccgggaggacaggttctgtccccatcccgcctcccacacaaagggtttccccggagccagaggaggaaacctggccttcctgcactt cctccttcgcacattttaaaccatccgaggatgcaactgggggaaaagatttgcagacaaaagagctgggggtgccggcaacagctg tttgggccggaaagcctggccccggctcagaccccggctgggtccctgcgctgtggcgggccggccgcgacccccgccccaccc aactccgccccctgctgcccgctgtctccggagcggcggctgtttggggctggctccttttctgggcccccacctccctgaagccccc actgcccctttcctccttcctggggccctgggagccagggcagggcccccgtgcagctttcagcagctccagcccaacggctgcag gcgctggggaagaccacagctcaggtgggaccgcaggtggtggctggaggcagccatcccacatcgcagcccccaatctcgtggt tttgtgctccaacggtgaacaggcctggtggtgctgggctgtcgtgggccccttggccccctcggtgcctgcatccctctctcctgtcc aagatggggaaggaagccgggagcagaaagggaagaaagacccagcggctgcagggcgcctttcagggcctcctagccaccgc agaggctccagccagctgtttgaacaggggttgggacacagggtcctggcaggtttgaggagcccggggccttccttgctatagctc tttgttctgggggatttggggaggccagggaaggggctgtgagcaacgaggttccagcatcttcccaagcctcctccatcccccagg gctgggctgcttggccgttccagtgggagaggacccgaggtgggaccccgagccccctgcccagctcatttcttagcagccggtgc ctcctggaaggtgggtggtgcccactgggattggggaaacttggtctagcctctacccaagggaggggctgaggtcccaggactcc ccctgccccagaagccttggtccgggctagagctgggctcgttggcctcatggaagcctgccttgggaagcatccccacccaggtct ggccttgtctccccgtctccagtagggggtcacagtgtccccccagccagtccacatatggccccatttgcttgggcaggccgatggg tgggcctgtctgtgtggcccaggtcacctggtggggcccactgtcagtggcgtaggtagaaggtggtcagccttgctgggtcttagca gagcacggcaggatctgggtctggggagggcgtgggggtgcccctgccatcacaggagcaggcagagtgggagccctagactc ccctctgggcagaaagccccttgaggctgggggcaggtcctgctggtgagggaggggtcccaggggcaggggcaccctcaggct ggccgcctagcgattggctcctgcaggacgtggccacgggctccctgggagcagcagctgttgggggtgtctggggagaagaccc ccatttattgccctgtgagggaccacactgctgccaggggacccttgggcttctgtggcaggacagtgggccgggattggcagtgag gcccaaggagggcaggtggggctggacctgtggctctgctggggagcaggtgggatgtgttaagatgctgatttcagccgggcag ctcctccccttcccttcccagggagccggggtgtgcagggccgttccttcccctctgggaagtgcagctcctgtgcaccggagaggc ccccgcactgtccctgcccgtcccaccaactgtccctgcccgtccctccactgtccctgcccatccccctcactgtccctgcccattccc cgcactgtccctgctcatccccaacactgtccctgcccatccccccctcaccgtccctgcccatcccccccgcactgtccctgcccatc cccccccactgtccctgcccatccttgccttgggaggtgctgtcacctggtggagccatgggaagcacctctcccacccagggtctct ggttcctgtggcgggtgcagagccagccacatcactgacaccccagctgggttgggtccagctgaccccacacccccacccctctg ctcggcctctgcctgtgcccccatccctggccctgcatgaccataaactccttgagggcaggaagtgagtgagtcctacccgcactga gtccctctcacccctccttctcattttggggcggagggaactgtcttctgtcccctgggggtggcagatggccgcttagcttagaaagg aaaacagcacaatttagaattcgtcctgggtgaaaggtctcggcagcatctgcctcctgctgctttctggagtcgcatttccatggagcg gccctggcagggagggtgggagtggcacagtggaaatactgaggcgggaacaaagtggtggcggtagaggtggcgggagcgg cacagcaggcgccggggccacttggcaccctccccccgcagccctcctggagtccccgcccgccgctcacctgcggggggccca gataggacaggttaccccagcgccgccaggccactgagcgtgggggatgggacaggcaggtgccgggtgaccctaagagggac gcacggaaggggagtccacgggggcacacctggcgtgggttgggtctcctccgtgggagcccgagagctgggtggcttggtgga cagggcagctctctcgtccagtcgggggagactggcgttccctatctcaagcctcactttttagccccagaggggtctccccaccgcc cctgataggaggagatgcggggacggtggaaggtggggtgcgggtcactcatggaagccctgaggatccccgtgggcgcggca gctgttgggtgccacctcactcggccaagccctggcgcccacctcgcggcccacacccacctggccgccgggacagcattgaaga cagagtggcccgggtaccgggggtgtggccagcgccaaagctgtgaaggatttgctcacaggctggtggtcctggccatggccatg ctgcaggcctgaggtccctcccaccttctcagggacgtctgggtgctgtgcggagggcaggtgttcgccgtctgtagggtgctccag ctctgtggccatgtgggagagaggtacagactggggcgggcacaggctggaggctgcccacagcagcagcgggagactgggcc caaagctgccccccacacacagacccccacacctggctcaaaactttctccaggctcggcccggttccccccagctagcgcgtctca cggaacctccaagccgtggctgtggggccgtggcaggggccttccctgtggtgtccgatgcccaggctgggagtgggggcggac aatgggccctctcagcctgcattcccccacgggagcctcctcaggcctccggctggggctcctgccccacccgctgggagcagctg ccgccacgtctgacacctgctcccagccgccctttgtccgagcaccgacgcctgatttatggggcgtgttttcctcttgctggcccggc gtgatacagcctcgttaactgggatgcgtccggttgggcagattctcccacagtcctccacgttggggtccgcgctcagggtgggga gtggggcccggtgtccgggcagagtgtcgtggtccattcagggacagccaatgtcctgtgacctcagggcacttggtgggagactg ctctaggggctcggacctccagcgtctcccctctggggtcccctaaagctgtgggatgggcctcccctgcccctcctccccgtcttgc ccagcaggtgagcttttcccgctccttggagcctcgaggggccgggaggcaggggaccagcttgcatgggccaggctgaggagc ctccagccccggctttgatgaccgagcgccctctgtcctgtacccaccccctgccgacttcagcggctgggaaagcaaaaagggcct tttctccaagcgggagctgtggccaactcccgcctgacaatggggcatttatgtggagggagggcgcccctcctgtggccacagccc agcccagcccagcctgtgcactccacgggttcccagcggccgctccacctcggggaccggccagcctgtaactggatctgcatgca gtgggaaccacgtctgccctcccaacaggtgcccgcagaggcaggtctgtcgctgcagccccggagtgaccggccacatgggatg gaggagggggagcctcattccggggctgtgcccgtcgctcactgggctggcaggagccgtagcaaggcctgagtgaattccccag agcccctttggaaggggtgacatgtgttttctttaagccagttggtttgggacttttggcttctggcaacagatggtcggccagagtggcc cttttgcgttctctctacccaagccgtggtcacgggtaaaagccttcggggaagacagatgcctcccgggcctttgggacaggggcgt ggctggctgctgagaaacgcacaggaacccccacccccagcctccacaagcccctcggggtaaggcccccccaggccactgcca cacatagcacaggtttgattggagacactggggtgggaataagcgggagaggaggcgaggacttggggggccggtgggggtcct gtgggaagccggcagcttcagggccggtcctggggcaggcggccgccgcagccccgcacccaggtgggctccggaaggcggg acggttccctctgtacatcccatctggaggaggccatgcccacccagggcctgagccgtgaggctgctgccttcccgggccgctgg ggccgctgaggctggtggcatgttctgcttcagaggcttcaccactgggaagagcggcgcccggcgggtcccagaccttttcccgg agcgcaaggccaggaggtgactggggccggagatttatgaccacagcaggtggctggggggggcaggctctgcgcctgttgccg cagggaacaggtgagaccaggcctgggtggagatgcaggacccctcccctggctccccttgttctctctgagcccagactgtgtgtc cggggtgaggggcagcctttcacccaccttgacggggttgcagagacctggcagagggacctcctcctggcaccttggtttccccg gctgtaatgtgggtgcttctgctcctggctgatgtgcctggatgtgagttatgaagactcaatgccgaaagggaaacgtgtctgggggc tgagcgtgggcagggaacgggccagccttcatccccagtgcgatcttgacatgagatgtcccatccccattgcagtctcagcctgag gtgtcccatcagaaaggcttcctggccgggcgcggtggcttacgcctgtaatcccagcactttgggaggccgaggcgggcagatca cgaggtcaagagatcaagaccagcctggccaacatggtgaaaccctgtctctactaaaaatacaaaaattagctgggcatggtgtcag gtgcctgtagtcccagctactcgggaggctgaggcaggagaatcgcttgaacctgggaggcagaggttgcagtgagcctagatcgc gcccctgcactccagcctgggcaacagagcaagactcaaaaaaaaaaaaaaaaaaaaaagaaaagaaaaagaaaggcttccacgt aggaaggggctgatccacactttccgtggctgccatcatgttgacacctgaaggttggaaaaaccacggcctttgttactgcccaagc aatgcctgttcgctttagcaaagctgggaagaaaagcagataggcctgcagacccggaggctctacggagctaatgcacccggctc ctctccatggctctccgcgggtgtagacccagacccctgcccgggctgtggagtccaagctgggttctgctggaacatcctagtctcta gcttgttccccaagttagcagtcatttgtcttcttctttctttctttttttgagtcttgctctgtcacccaggctggagtgcagtggcgtg atctcggctcactgcaacctctgcctcctgggttcaagtgattctcctgcttcagcctcattcagtggcattcagtacattcccaaagttgt gcaatcgcggcatgtgtctggttccagaacattttatcagccctaagggaaccccgctaagcagccgttgtgcccgtgcccggggtgtgggg ggaggcggctgtgcccatgacaggttcccggtgctgagtggcgggggcgctggggaagcaggtgggaactaactgccctggcac atctgccaacagctgtggcggggccttcgtgggcccgcgatgccaggaccccaacccgtgcctcagcaccccctgcaagaacgcc gggacatgccacgtggtggaccgcagaggcgtggcagactatgcctgcagctgtgccctgggcttctctgggcccctctgcctgac acccctggacaatgcctgcctcaccaacccctgccgcaacgggggcacctgcgacctgctcacgctgacggagtacaagtgccgct gcccgcccggctggtcaggtgagggaggacccacagcctgaggggtgggcagacccaggcccgccgccagctgcccaggcaa cttgaggtacttggcgggatccaggcccgggtctctgacccggaagtaattgggagcccctgccccagggtccctgcccccctccca gacctggaatcgcctcgttttcagctgcttgctctggcccaactcttgctgtccccgcttctgcgagacaaggggagcctctgtgtccag gccgcccctgcccccttcctcctgccgtgcactggccctcacctgttagggtgaagggtgtctggaggtgtcagctctgggaagaga cggcccaggctcaggcctcttgggagctgtggtccttcatctgccaaaggcgaccggtctcacggcaggtcctgaggatcaaatgtg atagcacctaaggtggagcccaggctggcacctccaggtggcctgaagggagggaaggcccggcctcggggagcagtgaggcc agcacgaccctcttgtccccttgtctccagggaaatcgtgccagcaggctgacccgtgcgcctccaacccctgcgccaacggtggcc agtgcctgcccttcgaggcctcctacatctgccactgcccacccagcttccatggccccacctgccggcaggatgtcaacgagtgtg gccagaagcccgggctttgccgccacggaggcacctgccacaacgaggtcggctcctaccgctgcgtctgccgcgccacccacac tggccccaactgcgagcggccctacgtgccctgcagcccctcgccctgccagaacgggggcacctgccgccccacgggcgacgt cacccacgagtgtgcctgcctgccaggtagtgctgcccgctggccggggtgcacgagcccctccccggctgccaggcccagggg gtgggaacggggcgcctggtgtaggggcagccctgggagggcccatggcggggagttgggaaggcgggatggcgagttcccca gactgcgtgtggtgtcggggggacgcgtctggagcggggtgtgcgctgggaggaggcttgcacagggtccaggtgccagcccgt gtctctggagtttcccggaactcacattcgagctgtaggacgccctgcctccccccagtgcaggcaccctgacttgcgcaacccccag ggtttcccctgcagtggggctggggccggagagggcgtgggagctgcgggggcaccatcacttcccaagccatggcccttcctgc catcctgctggtctcctgcgggtgtgggtgacaggtaggcgcagccgcctgcccgtgtccccagtgctccccgatccccgtgttgtac gtaggagcctctctgctgtagttcttgtttgagtaaattttgcagcagctctaaaaataaggaagagttttgaaaacctccagtctcggccg cgcgtggtggctcacgcctgtactcccagcactttgggaggccgaggtgggcagatcacaaggtcaggagatggagaccatcctgg ctaacacggcgaaaccccgtctctactaaaaatacaaaaaattagccaggcatggtggcacgcgcctgtagtcccagctactcagga ggctgagggaggagaatcacttgaacccgggaggcggaggttgcagtgagccgagatcccgccactgcactccagcctgggcga cagagcgagactccgtctcaaaaaaaaaaaaaaaaaagtgaagagaaaaaagaaaacctccaatctcagctgcacagaatggctgt taggtgggaatggggagagggcattcaggcaggggtggggctggagggtgcacagggtcctggctgacccacgggtggcccaa gggctgccccacccccttggaagccccaagctggcctggcccctgcaaaggggaagctccctggctgcccagtcagctgagccca gccacctctgcgtgcagacaccagacgtgtgcgtttcctgcaagtcactgggcgagcgtggggtggggacagacactccctggcca gcttggatctctgcagggtggggcgcagagcccttctgctggtgggggctcagatgccttcctagcagcacgggaccctacctccag tcacacggattgaggggccaagctcttggggggccgtggttcaggctagggcaggtggggtgtgtgcggccttgtccaaggccacc tgtgtgcagagctgatgcccacccgccttgggcagggtgggtgaggacctcctggtgaggcccagcggcagagatgccaggggtg ctgggccggctcagagccactgcccgtgcaggggcagctcagggccctggggagtgggcagggagggaaggggaggggctgg tggggttcactcacttgagtgtgggtagatctcagaggctggggtgctgggggctgggtcacccgtccggtgaggggtgtgctcact gcctgctgtggagtctggccgtttcagggtgagatgaccatccgggggcctccccaaatgactcaggcctgccagaccccagcttca gactgtccccggcggcccctgcacccaggccccctgaagggccctcagccacctctgggtggtcaggagcccaggagacagctgt gtgtggagtcacggggaaccgagatgggggcggccctgagctctgcaggcccgggaactggctgggatttggggtcgcacagcc tgcctggcacgtcagcactgtctcctcgtggggtcgctggccgaggctacgtcgcagagggtccagctggggtccacggggctctc gctggcttcccgccctctcccccactgtgtgcctcagccagtgctttgcagggcagccctggggtcgcaggcccagccaggggccc catctgcctcggagccggggtgcagggccccacacatgagtgggaccgggactcaggctccaggctctgaattttttttttttttttgag acaggtctctgtcactcaggctggagtacagtgacgtgccctcagctcactgcagccttgacctcccaggctaaagcgatccttctgcc tcagcccctcaagtagcttgggaccacaggccggcgccaccacgctttgctacttcttgtatttttttttgtagagacggggtttcgccat gttgtccaggctggtcttgaactcctgggcgcaagcgatccacccacttcagcctcccaaagtgccgggattccaggcgtgagccac cgcgccagggctggggtctggttttgggtgctccccaagctgatttgcattttggattttggttgtctctggctgcctgcttttgctccggtc cagcagacagacagaacggacggggtggtggcggtggggcaatgtctcagagggaggggccgagcaggtgggagctccaagg gcaacgggggttcaaaggcatctcgggagggcggcaattagtaaccggctgcaggtttggctgttgccgaacgcgcctttctgggtt gatcattgaaccagcgggagttgagtggattaatagctaactggctgccttggaggcctgggtggggccatacctcgggggagggg gccctggcagcctgtggaggaggagggggaggaggaggtgggcaccagagctctgccccagggaggatgggccatctgaatct ctgcagcttccctggggctgcgaggggcccaccctgccccagccagcagcctgctggccccatgtccccctgctgctcagagccca ggccccagtgtcagcccctgaagatggggtgtgcggcccccctgtggagacattgtagggagctctgagcatgcaggcggcaccc aggtgcaggagctgccggaacctcttaaaagagacccaagggtttttgtgacagaatcttctggaaccctgtgttcctgagtccctgttt agccagtcctgtccaaaatgattctttcaagcagggcgtgtccctctgggcactggagtgaggcaggggacgggcacagaccccgg gggccccagggcagggccgtggaggccaggctcttgtgtccagagcagtgtgtcgggggtccaggcaggagccggactggacct cagggaagaggctgacccggcccctcttgcggcaggcttcaccggccagaactgtgaggaaaatatcgacgattgtccaggaaac aactgcaagaacgggggtgcctgtgtggacggcgtgaacacctacaactgccgctgcccgccagagtggacaggcgcgtatacgg gtcgccgcagggcggggtagccggggcggggctgctacccacttactaaactgcactcacaggcatagcggggagcagccgaga ccctgtcccaggccaggcagactagggttggccgagaaggacaacatgatgggggccccacttcccgtggccagggtctggtgcc cctcacctcgggggtcagcacagacagggccctggttggtctgtgtgggccgtttgcaacctggctccaggcagcccctgtgcccag tggggtgtcctggcattgagggccttcagcaccccactcaggccacgcttcctgggccagtcacagggatgcctggatgaacagac aggccctgtgttcgggaattaccctctggtggggtgacgggcaaaggtgcaccccgacgcagcagtgtccacagagcacaaagag gaggcaagactccatggtgctgggcctgcagaggaaggagtgatttaggctggcggtgggtggaggagtcagcccaggaaggctt cctggagggggcggcattgccaccctgcgtcttaggggacagggagctcagggagtggcagctgcccggggccgacagctcctg ttccctgcaggtcagtactgtaccgaggatgtggacgagtgccagctgatgccaaatgcctgccagaacggcgggacctgccacaa cacccacggtggctacaactgcgtgtgtgtcaacggctggactggtgaggactgcagcgagaacattgatgactgtgccagcgccg cctgcttccacggcgccacctgccatgaccgtgtggcctccttctactgcgagtgtccccatggccgcacaggtgagtgccttgaggc ccaggtgggcgcagggggctcacatgggccaggcctgagctgtgtgacctcacccagggactgtggccctcctgcacccaggata acctgaaagggcctttctggtcagggaggccggtggtcgctgtgagtcccccggaacgtcccaagtgtcacgggatgcccgacgg ggtcacggcgggcagctcctcctggggtgtcagtggggttggaccccagccgtgggtggtgtgccatgcctggcccaggggccgt gcccactgaccgccgctgcctgcccaggtctgctgtgccacctcaacgacgcatgcatcagcaacccctgtaacgagggctccaact gcgacaccaaccctgtcaatggcaaggccatctgcacctgcccctcggggtacacgggcccggcctgcagccaggacgtggatga gtgctcgctgggtaggtgccagcacagggggtgcggccaggtgggggtggcagcctggccccagaaccgatgagaagtcgattct ggcttcagggggtgcccagttagatgggggatgggaccctgccagtccgatgggggtggtgtgcagtgaggtgttgcaggggcca gggtgcctggagcagcctctcacccgtgtgccctcgccaggtgccaacccctgcgagcatgcgggcaagtgcatcaacacgctgg gctccttcgagtgccagtgtctgcagggctacacgggcccccgatgcgagatcgacgtcaacgagtgcgtctcgaacccgtgccag aacgacgccacctgcctggaccagattggggagttccagtgcatctgcatgcccggtgcgtcggccggggccagggcgggaaac cgagtcgaggctgggcagccttggaggggcagccccgggaacatctgtgggttgcttccgctttccccagcctccatgccttctggg cccacagcctgcacagggcatggggaaactgaggccaggccacagggccagcagagcacgggggcagtgctaggcagatgag ggtgccgggccaggggccacgggctgcaggggagcgggtgcgcagggggcccgtggtggctgggacactgggctggaggca gggttcgtttctgtcccaagtccacagctgtgcccagtggggcactggggccgcgcgtgcccccctcactgttgccccaccccacag gctacgagggtgtgcactgcgaggtcaacacagacgagtgtgccagcagcccctgcctgcacaatggccgctgcctggacaagat caatgagttccagtgcgagtgccccacgggtgagggccacccccgccccctgcccccgggtcgtctgcaccctggcctcctgagg ggtgcctgggcgtgggttgtgtcccctgcccccgggccatctgcaccccggcctcctgaggggtgagggccgcccccaccccctg ccctcgggccgtccgcaccccggcctcctgaggggtgagggccgtgggttgtgtcccctgctcctgggccgtctgcaccccggcct cctgaggggggcctggccgtggattgtgtcccctgcccctgggccgtctgcaccccggcctcctgaggggtgcctggccgtaggtt gtgtcccctgcccctgggccgtctgcaccccggccttctgaggggggcctggccattggttgtgttcagattcccaggagagcgagg gttttgcctcatgagtggatgggagtgttttcagacttccccgaaggaagggcagggcccagtggggagtgggagcttgcccaggg gtcgcggtggagcccagggccaggagatcctccttgatctgggtgcagccccttctgcctggagagaagggtataccaggagctgc agtgcccagacagggaggaggctccagcctggctttctgagggctcgatgctgggagggaggccagcctgccctgtcctgccatgt cccctgcccaacggcacgccagcgacaagggtatgcagagaaccacgtggggcaggttggcctgcaatggagatgatggctgcc cggggcggacttggaggaactctcaggggcctgggtgaaaggtgtttccatctgtcccagagctgggggccggggcgcagagccc agggaggcgttgccagcccaagcctaggctcccaagacatagattacccgtcccagacactggagcgaggggagccccattctca gcccggctccactgtagccatagcaacccagtcggtttgggaaaaaggccccttctgtgggagcgagggcgtgtggtgctggggga ggggctttgatccggggagcgggcagcgggaggcagggagagctggagctggctgagctcaggctgagtgtgccctggggagc ccagccaggcgctcactgctggggtctggccaggggtccctgaagggccatagtgctgttgcacatgactccctcccctcccctccc cggccccaggcttcactgggcatctgtgccagtacgatgtggacgagtgtgccagcaccccctgcaagaatggtgccaagtgcctg gacggacccaacacttacacctgtgtgtgcacggaaggtgcgggctggcgcccaccagcggggagggactggggacgggacag ggcactcagggcagtgaaactgacaaggtcatggacaccttggtctgggccacctggtccagaggccgagagcacagctcagccc caatcctgagtaggtgggaatgccacgctcggagccctcctcaggttggcacaggtgaccccaggtctggtcatgggtgtcccggg ggccgccagtcctaagtcttcctgtgcccgcccctcccacggtccagggtacacggggacgcactgcgaggtggacatcgatgagt gcgaccccgacccctgccactacggctcctgcaaggacggcgtcgccaccttcacctgcctctgccgcccaggctacacgggcca ccactgcgagaccaacatcaacgagtgctccagccagccctgccgccacgggggcacctgccaggaccgcgacaacgcctacct ctgcttctgcctgaaggggaccacaggtggccggccaggcgggtggccggcggggggccagtgggcagggcgggcctgagga ctgaccgacacgtgccaccccttcaggacccaactgcgagatcaacctggatgactgtgccagcagcccctgcgactcgggcacct gtctggacaagatcgatggctacgagtgtgcctgtgagccgggctacacaggtgagcggccctgcacgtgggggctgactgcactg tgctcagaggtcaaggtcagccactctgccctggggctgctgaggggttgggtgagggctttggtgctgcccacctgggcagaccgt ggtctgcagcagagattgcagcgggaccagggctataactggcagcacgatgggtccccccgacccctcttgtctccggtcagagc tgtcccaggccgtgttggcagagtggcccagcaggccaagagggagcagccagccctgaggccaggcttaagtaccttctgcctct gtggctcaccagcacctttgagcaggtccctctgcctctctgggcctcagattctcatcagtcagggctgcagtagcccctgcccctgg ggcccctgggaggatgacttgagtgggcactggtgcccggggaggctggcacatggcacattgtgatccaagatggtttctgacacc tggaaggatgtggccagaagggtgatcgccccggctcaacagacagggaaatcgaggttgacgtgggtgggaccccctgggcgc tgggcctcggagtctgacccgccctgcccttagggagcatgtgtaacatcaacatcgatgagtgtgcgggcaacccctgccacaacg ggggcacctgcgaggacggcatcaatggcttcacctgccgctgccccgagggctaccacgaccccacctgcctgtctgaggtcaat gagtgcaacagcaacccctgcgtccacggggcctgccgggacagcctcaacgggtatgcggcggggccgatcatggggacacat cagtcctaaaccctgggagctctgctgccaggagggtggcacctgcacagagcttgagatgggccagaaacgggcctcggacgg ggctgggtggcagggagctcccctcggggacgcacatgcctctgggtcctcagtcagactacagtccacacccagcagctgtgtgc tgcgtgtccttgtgggctgcatggtgtgctcaggacacaggcccacagtgaccctggaacacgtttccatggtaacagccgcaagtgt ttctgatgcccatgatgtgcctggcagcactctgcctggatgggaccatttaaatcagcagggaccccattgccctcatttggcgcaga gaaggcacgtgctttgttcggggccacacagcgggcaagtggtggggggcttttcgcagcaccctactgccctgcacttggcatggt cccctcgctaatgaaccaaacccctgccgcagtcttgggtatgggaagcgctgcggcccccacttttttctttattttgagacagagcctt gctctgttgtccaggctggagtgcaatggcatgatctcagctcactgcaacctccacccctcaggttcaagtgattcccctgcctcagcc tcccgagtagctgagattacaggcatgtgccaccacacctggctaatttttgtatttttagtatatatggggtgggggagtttcgccatgtt ggccaggctgatcttgaactcctggcctcaagtgactcagccacccccgcctcccaaagtgctgggattacaggtgtgagccacagc gcccagcctgcccccactaaaggactctgcgagtctgagtggatgggcacctccgccagcccatagggcattgcagacccgggag tgcccaggccccggccgtgctgctcgggcctccctcgacctgcagtgtggtcccccttgcaggtacaagtgcgactgtgaccctggg tggagtgggaccaactgtgacatcaacaacaatgagtgtgaatccaacccttgtgtcaacggcggcacctgcaaagacatgaccagt ggctacgtgtgcacctgccgggagggcttcagcggtgagtgggctgcgcgtttctcagtgcagagggccgccctcgagtctgggga gctggagacacaggatcgggacccaggtccagccagcaccatgcatggtgtctccctcccgtggcaaagccttagctccaggcctc tggctcttggagatgaggaggggcctggggtggggagcaggcccagggctgctgttgtggggccctgccaaggtgcctgggagtg ctggacatgccgagtgctgtccccttccctccaggtcccaactgccagaccaacatcaacgagtgtgcgtccaacccatgtctgaacc agggcacgtgtattgacgacgttgccgggtacaagtgcaactgcctgctgccctacacaggtgaggggtgggtggggcctgtgctg gagggggcggggcctatgtgggaggggcgggacctgtgctggagagggcggagcctgtgctggagagggtggggccaggggt gggggcggcctgggaagccgtgactggagggtaaagtggtggctttcttggggcggggccttccagaaggttctgcgcctcactga gtgccttaggcccctgcagtatgtgggccttctcccgggggtggggttggcagattagcaaagatacaggatgcctgggagaaaattt tcatgcaagcatgtgttttacctggcaagcctgccgcaagacacaggtaagatgcaggtgaatccttagcagggccgggcctgggtc actgggctccgtgctcccggcgcccaccctgctggggtctctgggggccgggcttccccacccccgccagccggacctgagggat tcctttgatggcgggctctctttgtgtgagcggaacaaggagccctctgtttgggctgagcggggagggggatgttcccccaactgtc cagcccctgctatgaacccagccaaggggatcacaggaaatgatctcctctagaaaagacaaagaagagtccacaagcaccgggg tcctggcgctccccaggagcgggcagagggtgctgccacggggcctggtcagggagggcctgcctgggccaggcagccgggct ggaggtcccacgcctgcggttcccatgacatccagcagcctgtggccagaacgctgttttctttgaccgaagttaaggacctgggtttt ctctcctgtgtcagtcagtgaggaccctccctccacggctgggcgcccctgtccctttcagacacgtcgatcgggccggcaggagga gcggcggtgctgacccgcttcctatctgcccgcttcctgcttcctttcgcacaattgtcggaaactctggcgcagttcctgcctgcccgg gtcaccgtggaaaccaataggaagagaggccctggaaagtcgccccagcaggccagggagataatgggggacagggaagataa cgcagtccccacggctataaacaggaagctcggccatgggcttgtgcagaaaaaggccctcggctgtgcacgtggttcccacatag cctttgccagacaccctgcgtggccctggaggtctccaggccgtgccccgtgccccgacacgcaggatgtgctgggggcctgggc ctgtcccctccatcgcagcccttgtttgttggcctcccgtgatgggcgtttgctgcctgtgacacggagccccaggattagtttcaggcc tcggtctcaggccagggcctttcctcgtcacccagagatgagggggcttgcgtccggggtgggcccaggtccctgcagtgtcgtgg agcccacccgactgaggccgcctctcctggtgcagtccttgttcctgggcacgcctggtggggtgaggcctaggctgtgccccgctg ccaaccgggaatgaggggtggcttctgagcgtgacatttgtgcagcttgtcttcagcgtccccccatctgtgctccactcctccctgggt cagggtcctggcacagccgggagcgctcaggggtctcggtgcacatttgcctcccggcgggaccagcagacggcactctgatggc ggaaagaccagcaggcggtggccgatttgggagatccctctgggtgaggctcccggggtcacgtgtgtctccttcccgcaggtgcc acgtgtgaggtggtgctggccccgtgtgcccccagcccctgcagaaacggcggggagtgcaggcaatccgaggactatgagagct tctcctgtgtctgccccacgggctggcaaggtgaggctggccagggcccggtgagggctgggatgggaggtcaggatgtctgcgg gacacaggcagctcccaggcaggctagatgagtctttgaagaggagctggtgggtgctgaggaggccctggtcggagaagttctg gaatcaggaattgacctgggagcaccgttcccaactccagttcctgtgaccttcttaggccaaaattaggggagaggggatggtcctg gggtcacggaagcctactcctgggtcgggagaggcactgtaggtgggtgggccagcctgggaagggcctggagggccaggggc cgctggtgaccaaccggcctcctcctgccccgcagggcagacctgtgaggtcgacatcaacgagtgcgttctgagcccgtgccggc acggcgcatcctgccagaacacccacggcggctaccgctgccactgccaggccggctacagtgggcgcaactgcgagaccgaca tcgacgactgccggcccagtgagtagccccgcggctctggcctcctccaggaagctctcaggcctcagttcccccgggcagggttg gtgcgcatggtgctggccataagcacccagggaggccggagtgtggccggggagggttgggtcaggtgtgggggatgtgctgag ggatggccagggtggttcaggagggccccagagccggcttgctcctccctgcacttcgcggaagagtcacgagagcccctcccac ggctcttcactgagccgaggatggctcgggcccggcctgcactcccgcctggctcatcgggccccgctgcaggccagggctctca ggcctcccagccctgcttcacagaggttgaggttggcagaagccgggggtcttgctggcatcacctggcaggcagaggcaagaact gtctctcctcccctgctcggtctgtgaagcctgcaaagctgccccctgccctggagcttgaggacaggagcaggtggggagagaga ccccaagcacaggagacgggtgtgacgcagcctgtgggtgctggggctccccaccagacacctttgtcacagggctgcttgccgca gcctcggcaacgaaaggctgggctgtccccagccatgcagccttcccggccccctcccgcaggtgtgggtttgtgcctgcccctcac actcacccttccgtcctctcccagacccgtgtcacaacgggggctcctgcacagacggcatcaacacggccttctgcgactgcctgc ccggcttccggggcactttctgtgaggaggacatcaacgagtgtgccagtgacccctgccgcaacggggccaactgcacggactgc gtggacagctacacgtgcacctgccccgcaggcttcagcgggatccactgtgagaacaacacgcctgactgcacagagaggtgtg cggggctcgagtgaggccgtggaagggaacgggcggtgcgggccacacgcagtagctggcagcctggcacaccatgcaggcgt ccgcctaggccggctgggcacttagcgctggctgcattgagtccagacattgttcattgtaccgacctgtccctcctgagagggccatt gtgacctcctcctgtgtcccgcaccaggagcgccggtagtctgtcctgaagaactggtgcgcttccttctattagacaacgagaactct gtccgttcttgtttcctttttgccgtgtttgccaggaagctcatttggtaaacgtcagtctcgtccctggtttctgacgtaattacctcccgt gttaccaggacggtttctggtttctccctcatttactttaaccaccctatgcccacgcgtttggtaaaaccactctcgacctgacttataaa gaaagcaggggagggaggtgtgacgtggtgtgagagccgggccccgggttcccactggcctccctgggtcagccaggctgccctggg cctgtcctggccatcaggcccctagggttgagcagaaggggaggtgctggcgaggcagaatctgctggagcggggacccaccaat gccctccactcagcccccgcctgcccacccccttgcagctcctgcttcaacggtggcacctgcgtggacggcatcaactcgttcacct gcctgtgtccacccggcttcacgggcagctactgccagcacgatgtcaatgagtgcgactcacagccctgcctgcatggcggcacct gtcaggacggctgcggctcctacaggtgcacctgcccccagggctacactggccccaactgccaggtgagtgcgccggccacaga ggtgcccgaaggaggggccctgggtgggtgcctacctgcgggaggtgggaacggtcaccccaggtcccactgtgtcggctcggg gtcacccccacacccccgggcagagggtttctggggatctgagcagccggtgaaggaacctgatgcggaagcagcaggcaccttc gtttcaatcccaggtttctggagccggggcaggagctcaggaattggggaattgaggaaaagtgttccttctagcaaaggccgaggg tggtctggacctgctgagggccctgagggagacccagcccaggctgttcctggtgtgggggtggtggggagtccaggggcggca gtttccacttctgtagaatgggttgcagcctgggttggagtaggccccttggcagatgtgcgttctgagctaccggggaaatggccgg ggcgcgggcacccagctgaccccaatctgtccccagaaccttgtgcactggtgtgactcctcgccctgcaagaacggcggcaaatg ctggcagacccacacccagtaccgctgcgagtgccccagcggctggaccggcctttactgcgacgtgcccagcgtgtcctgtgagg tggctgcgcagcgacaaggtaacctgctgtgcccacccagctcgggtcccagcccatcaaggtcctctgtgggcctgggcctcacct gtctaccaccccatcccccgcaggtgttgacgttgcccgcctgtgccagcatggagggctctgtgtggacgcgggcaacacgcacc actgccgctgccaggcgggctacacaggcagctactgtgaggacctggtggacgagtgctcacccagcccctgccagaacgggg ccacctgcacggactacctgggcggctactcctgcaaggtgggggtccctcctagggtaagggttgtggccggcacgagtgttgcc acacaccaggccctggctgggagctggcccagtggagaaaactgaacctgataggcccatgcactgttcagtctcattaggggagg gctggggtaatcagggtagactgcctggaagaggtggcctgtggaaagcctgaaggagggtacctatactgagaagtgggatggg gttttcccttcccctagattgtgtctgggcttggccaacacctaccctgaggccctcacctctatcctatgggacggggtccacccaccc ccaacaggcagtgactccggtcaccgaggccccgccagggtctgttgggctgggtctctctccaggtctgacaggagcgaggggc ccgtggcttcgctggacctgagggcagctcatgtggccctgtcagccctcacagtggggtgtgggagcactgcatcctggcgccgg ctgagccgaagggcccctcgttctgtcgcctgcacagtgcgtggccggctaccacggggtgaactgctctgaggagatcgacgagt gcctctcccacccctgccagaacgggggcacctgcctcgacctccccaacacctacaagtgctcctgcccacggggcactcagggt aagggccgctgcacggagggctggtgttggccatccatggccagggcaggggcagggcaggcaccccgggaccggccagagg catccaggaaccagccagaaactcccattttcctgtgtgaggccaaggccgactcacagctgcccagggaaagggcccagagcgg gggtcccagtgggaagggcgtctccacggcacccttgacacctgcctctcccgagtgtccgtgcagccccagacctgagcgctcgt cttccgggacggacacgcggcacggcagggccggggtgtggcgggcttgggccactgacgaaacctggccccgcaggtgtgca ctgtgagatcaacgtggacgactgcaatccccccgttgaccccgtgtcccggagccccaagtgctttaacaacggcacctgcgtgga ccaggtgggcggctacagctgcacctgcccgccgggcttcgtgggtgagcgctgtgagggggatgtcaacgagtgcctgtccaatc cctgcgacgcccgtggcacccagaactgcgtgcagcgcgtcaatgacttccactgcgagtgccgtgctggtcacaccggtgggtgc cgcgcccaggcgggtggggcgtgtggggcagcagggtgagcctctcactgccctgctcttacccctagggcgccgctgcgagtcc gtcatcaatggctgcaaaggcaagccctgcaagaatgggggcacctgcgccgtggcctccaacaccgcccgcgggttcatctgcaa gtgccctgcggtaggtgcaggggtgcagggaggcaggggcccgccaggggagacacctggagaggtccacgtgggggcctcg gggcgcagaccgggcagtgatcctcccggccttcatcctcctcctcaccctgatgtcttttttttttttttagtttcaataaatgattttag agacattttagatttatagaaaaatggagcaggaagtagactccctggggtggctgtcccctgcacgcagttcccctggttagcagcttgcat caatttcactctggatttcagtgatacattgttaccaacagcagcccttcctgtacctggggcttgcccttggctttgtggttgtgggtttgg gctgaggtatgacatgcctgcccaccctcgcaggatcacggcagagagtccctgccctaaagtcctcagcactccaccagtttaccc cttcctccatctcccgaccccctggcacccccgatctttctctgttggtagaatgtgtgtaaagggttttgctgctgggggcaaggttgca ggccgcctcccaggttagaggagagcggtggcactgctggccgagggctgggtgtgaggtggcgggggggcggggggtggcc caccccgacaccgtcctgtcttccctctcgggcagggcttcgagggcgccacgtgtgagaatgacgctcgtacctgcggcagcctgc gctgcctcaacggcggcacatgcatctccggcccgcgcagccccacctgcctgtgcctgggccccttcacgggccccgaatgcca gttcccggccagcagcccctgcctgggcggcaacccctgctacaaccaggggacctgtgagcccacatccgagagccccttctacc gttgcctgtgccccgccaaattcaacgggctcttgtgccacatcctggactacagcttcgggggtggggccgggcgcgacatccccc cgccgctgatcgaggaggcgtgcgagctgcccgagtgccaggaggacgcgggcaacaaggtctgcagcctgcagtgcaacaac cacgcgtgcggctgggacggcggtgactgctccctcaacttcaatgacccctggaagaactgcacgcagtctctgcagtgctggaa gtacttcagtgacggccactgtgacagccagtgcaactcagccggctgcctcttcgacggctttgactgccagcgtgcggaaggcca gtgcaagtaaggctgcggggctcatggggctgagggaggacctgaacttggatgtggcctggcttgggcccggaggccagcatgc agttctaaggctctgctcagggggtgcagggacgtcccccgcggctggccagtgggctggaggcaccggacggcgggtgcgagg ccccccgaggaaggcggcctgagcgtgtcccgccccccacagccccctgtacgaccagtactgcaaggaccacttcagcgacgg gcactgcgaccagggctgcaacagcgcggagtgcgagtgggacgggctggactgtgcggagcatgtacccgagaggctggcgg ccggcacgctggtggtggtggtgctgatgccgccggagcagctgcgcaacagctccttccacttcctgcgggagctcagccgcgtg ctgcacaccaacgtggtcttcaagcgtgacgcacacggccagcagatgatcttcccctactacggccgcgaggaggagctgcgcaa gcaccccatcaagcgtgccgccgagggctgggccgcacctgacgccctgctgggccaggtgaaggcctcgctgctccctggtggc agcgagggtgggcggcggcggagggagctggaccccatggacgtccgcgggtgagtgagacccggcgcccacggtcaatccc cgcaactctcctgggccctccccgacggcctccctgcccctcacggccggcgccatggcaagcagtactctccccactttatggtaa aagagacggaggttccgagaggacctgggacttcagggcctgcgcaggcaaggagtagaggcagcattccagctcagggccctg accccacagccacacccttttcctgggccactgccttcccctggacaggcggcactcctgtgcccagtaggtgattttgagattgagct gtgccttaggcactggatactacactgattaaaactcagccctctgccaggcgaggaggctcacacctgtaatcccagcactttggga ggctgaggcaggcggagcccttgagcccaggagttcgagaccagtctgggcaacatagggagaccttgtctctgttttttttaaaaaa gtattaaaagaagtaaaaaacaaaacactcagctctccaggggttcccacagggctgaacagccccaccccagacaagaatgccgc ttggtcatggcgtctgcctggcttgggctggagaggaggcaggtggaggtcctgggagggggcattgctgggccttgcagttggag gaggagctggtgggggtggggggtcccacggtgggagaacacaggcaggagcagcttgagtgcagggggcaccccacaaggc tcccgcccatgcctactcgatctggggcagctggacccaggagccaggttggttgtgcccttcgtgtgcctgaccctggtgggtttgc ctgtcagttctgctggcttggagcaatcctggaggtcagaagcatcatctcacaggctggatgggatcctgctcacgggaacccagtc ctggggagcagagctcacccccagccagcctcaccacacagcccaccaccgctgcacccacccccacctcacgcctgtgcttcctg cagggcctggggatggctcctgggggaggactgggctcctggcacatactctgtcctgagatgaggaaacgtgtctgtggcaccag caggagccagagagggtgtcagggcgggccagggagagcgcgttggtgggtatctgggatgagccgtgatcagcactggccgg agtcggggggctggcaccagtcccctgcagggtagctgctgtcagacctggcttcccaccaccccaggctgcctcaccatgtcctga ctgtggcgtcatgggcctcagtgtcctgcggcagcatccctggccggtgggcgggggaggaggaagcctcgggtcccagcccctc tctgattgtccgcccagctccatcgtctacctggagattgacaaccggcagtgtgtgcaggcctcctcgcagtgcttccagagtgccac cgacgtggccgcattcctgggagcgctcgcctcgctgggcagcctcaacatcccctacaagatcgaggccgtgcagagtaagtgtg gccccatcccgggaacaggctctgcctgcagggggtgccatccccccgtgccccagacacgctggctgtttgtgccagttgctaccc acgggtgtgagcgttgccgtccgagttggggtagggcttttctggaattttctgaatggcactccgcccccacctgcggcggtcacag ctgccggtggagccacctgggaacgagtccagccacgggaaagtgggtgcctgcttctctccccaccctttcctcctgaattttctttgt tgggtattatttcaaaatcattacggctttttttaaagaaaaaaaaagagagagagagaagaattgatcggtgtcatgtgaagtgttgaag tttgtatcttgaaaatccctctaaatcctttgtcttaacagctcagtgcgagtgcagcgatttgaagttgactaatcctccttccttaaaggag aaaaaagtaaaagccgtctccagatagagtcggctggtgcaggagagaatttagcgatagtttgcaattctgattaatcgcgtagaaaa tgaccttattttggagggcgggatggaggagagtgggtgaggaggcgcccggacgcggagccagtccgccgccccccggccacc agcctgctgcgtagccgctgcctgatgtccgggcacctgcccctggcccccgtgcccgcaggtgagaccgtggagccgcccccgc cggcgcagctgcacttcatgtacgtggcggcggccgcctttgtgcttctgttcttcgtgggctgcggggtgctgctgtcccgcaagcg ccggcggcagcatggccagctctggttccctgagggcttcaaagtgtctgaggccagcaagaagaagcggcgggagcccctcgg cgaggactccgtgggcctcaagtgagcggacgccgcccctgcttctgggtccccggtgggaggtgggacctggccgagcatcctc accgggagtctgcccctcccgacccagggcccacctcccacgccaggcccgggccaggggtctctgggggcttcctagggagct cgctcagcctcacttctcgacccctcaccccccaggcccctgaagaacgcttcagacggtgccctcatggacgacaaccagaatga gtggggggacgaggacctggagaccaagaagttccgggtgagtcgcgaggctcccgggctcctgggctcccgggcacctgctgc cgggctgccctgacaggctctgctcactccctctatgtagttcgaggagcccgtggttctgcctgacctggacgaccagacagaccac cggcagtggactcagcagcacctggatgccgctgacctgcgcatgtctgccatggcccccacaccgccccagggtgaggttgacg ccgactgcatggacgtcaatgtccgcgggcctggtaagggtgccagcagccagggcttccctagccccgtggcccacctgcctctct cccctaagccccgaggctggggtacagttgatactctggaaacttagaattgggggtgagagctttcatccttggggtgtttcccattca gagtagacgtgggggggtcctggagtcctcctgttcttccaccaaccccttcctggggtgataccgcagggccactctgtccctgtaa attactcttttctgaaaccttcttgagacatggaaagcgttgattttctttttctttttttttttcttttttttttgtttttgagatggag tctcgctccgtcacccaggctggagtgcagtggcacgatctcggctccctgcaacctccacctctcgggttcaagcaattctcctgcctcag cctccccagtagctgggactacaggcgcctgccaccacacctggctaatttttgtatttttagcagcgatggggtttcaccatgttggccagg ctggtctcgaactcctgacctcaggtgatccgcccacctcggcctcccccagtgctaggatgacagcgtgagcctccgcatcctgctgaagc attaattttctaactgcatgcttctggggtcctctttttcctgggtggattttgggtgccaggtcttgggcaggtcaggaagcagttgcccg ccaggagtttaagctggattcggctctgtccactgagcagcccaggcggacttcagctgccccttggtggctgtgtgcacggggcca ccaagtgctgggtggtgcatccacaccgtggccccttgagcttggggctgctgcccccctccccctgggctgcagctggggaccgg ccctccagactgagcacccgtctctgcctctgcagatggcttcaccccgctcatgatcgcctcctgcagcgggggcggcctggagac gggcaacagcgaggaagaggaggacgcgccggccgtcatctccgacttcatctaccagggcgccagcctgcacaaccagacaga ccgcacgggcgagaccgccttgcacctggccgcccgctactcacgctctgatgccgccaagcgcctgctggaggccagcgcagat gccaacatccaggacaacatgggccgcaccccgctgcatgcggctgtgtctgccgacgcacaaggtgtcttccaggtaggcagtgg ctgcctgtgtgcccacctgccctcctcagggccgcctggtggtctggggcagtggccaggcttacgtggccctgggagcctgaccc cgagcacagctgagtccgggacaactggtgcctccacctgggaccttcgcagtcagcgaggtgcgagggggagggcgtcgggcc catctgtgttctccagggaagtcaggcagaggcgggtctggcaggaggcctgggggatctgctgagtgaggcagcacctccccac ccccagcaaaacaggctccatcagggttgtgggccttgctcaaggtccaggttccactgctgcagcccctcgcagccccgcccctcc ctcaaccttggctgccggcgtagcctgtggcagtgagaagcagggtttagaggctgccgctcggtgcctgcagacctagggctcag cttgccggtgagctcgtggcaagaatggatttagggatttggatgcctgggtctccagggagtgtccctggcaggggctgcctttgca gtcacccctgctgcgagtccccaggctccaggcggccctggagcaagcaggttcagatgggcacagcccggggagttaccacag agcttctcatttcctgatttcttagctcaggtgacacattgtcatctcgcagaaataaatgtaggtgagcagaaagagcgtggaagggag ccccgtgcgggtttggtgtgtgcctctctcagctgccttctttgcacagatgtggaagttcccaggtgctttgcaggaatcaggccaagtt gccttttgcacccgccagtgagcaggggccattcctcctgccagatgccaagacccggctgcattacaagggctgcccaccccctct ctgggcagagccggacactagctcggcggttctgagacggctgtagggccgtgagcccggcttcctgagtgccagctgtaaccgcc gttgggggcagggacttgacctctctgttttgtaggtggtcatggcagctaggaccacagtgaggattaaatgaggccacacgtggtc acgatggatcccactgtcaccaaggggcctgtctgtgggcagcacagcccctgcccccatccgggcccctcctcaggggcagccc catggcgtttcgtcttgcccggccgtgccgatctcaggagggtctcgtctgtgtccggaagacagtggggcttcccgtccaggcgttc gttctgggcaggaggcatcggtgtacgtctgcccagcacccgcctgagcctctccctgttgcccagatcctgatccggaaccgagcc acagacctggatgcccgcatgcatgatggcacgacgccactgatcctggctgcccgcctggccgtggagggcatgctggaggacc tcatcaactcacacgccgacgtcaacgccgtagatgacctgggtgagcccacgggggcacggctgctctgtcgtggggcgggacc gccacagggacgggtgggactgggttgcctccagctggtctccaacctaccccatctgcttctttcacgcaggcaagtccgccctgca ctgggccgccgccgtgaacaatgtggatgccgcagttgtgctcctgaagaacggggctaacaaagatatgcagaacaacagggtga gcgcgaggctgggatgccaggggagacgtgagggctgaatccacagagaagtgagggccaaacccacggggcgcagggacac aagggcctgacccacagggtctcgagggtcgctgggcccgcatgtgggccccacccgcatgatgcgggcacctcgttagtgctcct gccttccttcagcccccgtttatggagcccttccaagtgcaggtccagctgtcaggacacggcggctcgcccctcagacccagcccta ccctcctgggcggcagtcatggcggggcacacagcctggcgtggggaacgctgctgctgccactgctgtgtcaccgtcacctggac ctcaccttctgtccccagctgtcaccaggcgggggtggggatgggaatgtggtgacgcaggtggtgcagattgagccagaacacgc gtggcggcagctccctgcggggcggggcctcttggtgtgttcaccaaggccaaggacctcaaggctcagaggaagaagtctcagg actatatccaaggggagccacccccagccctcatcctggccctgcagctcctggccctgcagctgctgttggtttcccctgggtgctca gggcacaggtgcagacacccccacctccctgccgccagaacccccacccccgcccccaactcctgctgcccccttggcatgtcag gctcaggcgtctctccctcctgggtgagggcacacagcgggcctgggcaccggggcatgttggcccaggcgctccccagtccgtg gcagcatggccccacagagactgggccccagagggcatcaaggcctgggaagccccttcccactcccacacagcagcctcaccc agacctgtgacgtgtccaccgcacagaggagaccccaggaaggagcttggtggccaccagggtggcttgatggccgtccagatga cgagctccctccctggtgccctcgccggtgcctctgaaccgctccaggaatttccttttgtgccttattgggggcagggaagagggcct gcagttggttagattttcagtggggtctgtgacccccccatagaggtggagccccgctgatctagggtagaggactgcacagatcccc tctctgggtgggtttcagaagatgtatcaaagccttaacatttaacaagagtcaggctaggtggttgcaggacgctggggtggggtcct gaggagcagcctgcctgcccccaccccgcggaggaggttgtactgctgcttcctctggtgatggaaccttggggagggtccccacg cctggcctggcccccctcaccggcccccgccctcatcccccaggaggagacacccctgtttctggccgcccgggagggcagctac gagaccgccaaggtgctgctggaccactttgccaaccgggacatcacggatcatatggaccgcctgccgcgcgacatcgcacagg agcgcatgcatcacgacatcgtgaggctgctggacgagtacaacctggtgcgcagcccgcagctgcacggagccccgctggggg gcacgcccaccctgtcgcccccgctctgctcgcccaacggctacctgggcagcctcaagcccggcgtgcagggcaagaaggtcc gcaagcccagcagcaaaggcctggcctgtggaagcaaggaggccaaggacctcaaggcacggaggaagaagtcccaggacgg caagggctgcctgctggacagctccggcatgctctcgcccgtggactccctggagtcaccccatggctacctgtcagacgtggcctc gccgccactgctgccctccccgttccagcagtctccgtccgtgcccctcaaccacctgcctgggatgcccgacacccacctgggcat cgggcacctgaacgtggcggccaagcccgagatggcggcgctgggtgggggcggccggctggcctttgagactggcccacctc gtctctcccacctgcctgtggcctctggcaccagcaccgtcctgggctccagcagcggaggggccctgaatttcactgtgggcgggt ccaccagtttgaatggtcaatgcgagtggctgtcccggctgcagagcggcatggtgccgaaccaatacaaccctctgcgggggagt gtggcaccaggccccctgagcacacaggccccctccctgcagcatggcatggtaggcccgctgcacagtagccttgctgccagcg ccctgtcccagatgatgagctaccagggcctgcccagcacccggctggccacccagcctcacctggtgcagacccagcaggtgca gccacaaaacttacagatgcagcagcagaacctgcagccagcaaacatccagcagcagcaaagcctgcagccgccaccaccacc accacagccgcaccttggcgtgagctcagcagccagcggccacctgggccggagcttcctgagtggagagccgagccaggcaga cgtgcagccactgggccccagcagcctggcggtgcacactattctgccccaggagagccccgccctgcccacgtcgctgccatcct cgctggtcccacccgtgaccgcagcccagttcctgacgcccccctcgcagcacagctactcctcgcctgtggacaacacccccagc caccagctacaggtgcctgagcaccccttcctcaccccgtcccctgagtcccctgaccagtggtccagctcgtccccgcattccaacg tctccgactggtccgagggcgtctccagccctcccaccagcatgcagtcccagatcgcccgcattccggaggccttcaagtaaacgg cgcgccccacgagaccccggcttcctttcccaagccttcgggcgtctgtgtgcgctctgtggatgccagggccgaccagaggagcc tttttaaaacacatgtttttatacaaaataagaacgaggattttaattttttttagtatttatttatgtacttttattttacacagaaaca ctgcctttttatttatatgtactgttttatctggccccaggtagaaacttttatctattctgagaaaacaagcaagttctgagagccaggg ttttcctacgtaggatgaaaagattcttctgtgtttataaaatataaacaaagattcatgatttataaatgccatttatttattgattcctt ttttcaaaatccaaaaagaaatgatgttggagaagggaagttgaacgagcatagtccaaaaagctcctggggcgtccaggccgcgcccttt ccccgacgcccacccaaccccaagccagcccggccgctccaccagcatcacctgcctgttaggagaagctgcatccagaggcaaacggaggc aaagctggctcaccttccgcacgcggattaatttgcatctgaaataggaaacaagtgaaagcatatgggttagatgttgccatgtgttttag atggtttcttgcaagcatgcttgtgaaaatgtgttctcggagtgtgtatgccaagagtgcacccatggtaccaatcatgaatctttgtttca ggttcagtattatgtagttgttcgttggttatacaagttcttggtccctccagaaccaccccggccccctgcccgttcttgaaatgtaggca tcatgcatgtcaaacatgagatgtgtggactgtggcacttgcctgggtcacacacggaggcatcctacccttttctggggaaagacactgcct gggctgaccccggtggcggccccagcacctcagcctgcacagtgtcccccaggttccgaagaagatgctccagcaacacagcctg ggccccagctcgcgggacccgaccccccgtgggctcccgtgttttgtaggagacttgccagagccgggcacattgagctgtgcaac gccgtgggctgcgtcctttggtcctgtccccgcagccctggcagggggcatgcggtcgggcaggggctggagggaggcgggggc tgcccttgggccacccctcctagtttgggaggagcagatttttgcaataccaagtatagcctatggcagaaaaaatgtctgtaaatatgtt tttaaaggtggattttgtttaaaaaatcttaatgaatgagtctgttgtgtgtcatgccagtgagggacgtcagacttggctcagctcgggga gccttagccgcccatgcactggggacgctccgctgccgtgccgcctgcactcctcagggcagcctcccccggctctacgggggcc gcgtggtgccatccccagggggcatgaccagatgcgtcccaagatgttgatttttactgtgttttataaaatagagtgtagtttacagaaa aagactttaaaagtgatctacatgaggaactgtagatgatgtatttttttcatcttttttgttaactgatttgcaataaaaatgatactgat ggtgatctggcttccactcccctctgctctggcctttggctccctttctgggagggaggcagggctgctatgctctgagggagccaggagtcg agggccccttctgctgggagagtgacggtgaggctgcctagtcctggcccacgggggtgtggggaccacgctgcctccagggact ccatggtgtctgcagcctgcctggtccaggccctttgtagggagatggacacacagcagcaagggggttgcagccctatgggaggt ggggctcgtgcctggggtgacacggctcccaggacaccatgcgtgagtctggccctcctggcagctcggggctcttctcctctcagc ctcggagggatgaaggtcccacccagccatcctggggacagcccctcagggagctctgcagcaggcagggggctgcatagggag ggccttgaggcagtggcatgacccctctacgggctggagaccacagggaggactctggcccctataggagggcaaagggagctgt cgaagcctatgagcagggcagcatgggtcgtggcagagtctggaaagttgtgtgatcctaaggaactggctgagcgaggcagagg cggcctggtcc Protein sequence: SEQ ID MPPLLAPLLCLALLPALAARGPRCSQPGETCLNGGKCEAANGTEACVCGGAFVGPR NO: 349 CQDPNPCLSTPCKNAGTCHVVDRRGVADYACSCALGFSGPLCLTPLDNACLTNPCR NGGTCDLLTLTEYKCRCPPGWSGKSCQQADPCASNPCANGGQCLPFEASYICHCPPS FHGPTCRQDVNECGQKPGLCRHGGTCHNEVGSYRCVCRATHTGPNCERPYVPCSPS PCQNGGTCRPTGDVTHECACLPGFTGQNCEENIDDCPGNNCKNGGACVDGVNTYN CRCPPEWTGQYCTEDVDECQLMPNACQNGGTCHNTHGGYNCVCVNGWTGEDCSE NIDDCASAACFHGATCHDRVASFYCECPHGRTGLLCHLNDACISNPCNEGSNCDTNP VNGKAICTCPSGYTGPACSQDVDECSLGANPCEHAGKCINTLGSFECQCLQGYTGPR CEIDVNECVSNPCQNDATCLDQIGEFQCICMPGYEGVHCEVNTDECASSPCLHNGRC LDKINEFQCECPTGFTGHLCQYDVDECASTPCKNGAKCLDGPNTYTCVCTEGYTGT HCEVDIDECDPDPCHYGSCKDGVATFTCLCRPGYTGHHCETNINECSSQPCRHGGTC QDRDNAYLCFCLKGTTGPNCEINLDDCAS SPCD SGTCLDKIDGYECACEPGYTGSMC NINIDECAGNPCHNGGTCEDGINGFTCRCPEGYHDPTCLSEVNECNSNPCVHGACRD SLNGYKCDCDPGWSGTNCDINNNECESNPCVNGGTCKDMTSGYVCTCREGFSGPN CQTNINECASNPCLNQGTCIDDVAGYKCNCLLPYTGATCEVVLAPCAPSPCRNGGEC RQSEDYESFSCVCPTGWQGQTCEVDINECVLSPCRHGASCQNTHGGYRCHCQAGYS GRNCETDIDDCRPNPCHNGGSCTDGINTAFCDCLPGFRGTFCEEDINECASDPCRNG ANCTDCVDSYTCTCPAGFSGIHCENNTPDCTESSCFNGGTCVDGINSFTCLCPPGFTG SYCQHDVNECDSQPCLHGGTCQDGCGSYRCTCPQGYTGPNCQNLVHWCDSSPCKN GGKCWQTHTQYRCECPSGWTGLYCDVPSVSCEVAAQRQGVDVARLCQHGGLCVD AGNTHHCRCQAGYTGSYCEDLVDECSPSPCQNGATCTDYLGGYSCKCVAGYHGVN CSEEIDECLSHPCQNGGTCLDLPNTYKCSCPRGTQGVHCEINVDDCNPPVDPVSRSP KCFNNGTCVDQVGGYSCTCPPGFVGERCEGDVNECLSNPCDARGTQNCVQRVNDF HCECRAGHTGRRCESVINGCKGKPCKNGGTCAVASNTARGFICKCPAGFEGATCEN DARTCGSLRCLNGGTCISGPRSPTCLCLGPFTGPECQFPASSPCLGGNPCYNQGTCEP TSESPFYRCLCPAKFNGLLCHILDYSFGGGAGRDIPPPLIEEACELPECQEDAGNKVCS LQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDG FDCQRAEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGT LVVVVLMPPEQLRNSSFHFLRELSRVLHTNVVFKRDAHGQQMIFPYYGREEELRKH PIKRAAEGWAAPDALLGQVKASLLPGGSEGGRRRRELDPMDVRGSIVYLEIDNRQC VQASSQCFQSATDVAAFLGALASLGSLNIPYKIEAVQSETVEPPPPAQLHFMYVAAA AFVLLFFVGCGVLLSRKRRRQHGQLWFPEGFKVSEASKKKRREPLGEDSVGLKPLK NASDGALMDDNQNEWGDEDLETKKFRFEEPVVLPDLDDQTDHRQWTQQHLDAAD LRMSAMAPTPPQGEVDADCMDVNVRGPDGFTPLMIASCSGGGLETGNSEEEEDAPA VISDFIYQGASLHNQTDRTGETALHLAARYSRSDAAKRLLEASADANIQDNMGRTPL HAAVSADAQGVFQILIRNRATDLDARMHDGTTPLILAARLAVEGMLEDLINSHADV NAVDDLGKSALHWAAAVNNVDAAVVLLKNGANKDMQNNREETPLFLAAREGSYE TAKVLLDHFANRDITDHMDRLPRDIAQERMHHDIVRLLDEYNLVRSPQLHGAPLGG TPTLSPPLCSPNGYLGSLKPGVQGKKVRKPSSKGLACGSKEAKDLKARRKKSQDGK GCLLDSSGMLSPVDSLESPHGYLSDVASPPLLPSPFQQSPSVPLNHLPGMPDTHLGIG HLNVAAKPEMAALGGGGRLAFETGPPRLSHLPVASGTSTVLGSSSGGALNFTVGGS TSLNGQCEWLSRLQSGMVPNQYNPLRGSVAPGPLSTQAPSLQHGMVGPLHSSLAAS ALSQMMSYQGLPSTRLATQPHLVQTQQVQPQNLQMQQQNLQPANIQQQQSLQPPPP PPQPHLGVSSAASGHLGRSFLSGEPSQADVQPLGPSSLAVHTILPQESPALPTSLPSSL VPPVTAAQFLTPPSQHSYSSPVDNTPSHQLQVPEHPFLTPSPESPDQWSSSSPHSNVSD WSEGVSSPPTSMQSQIARIPEAFK CCR5 SEQ ID gaaaatccccactaagatcctgggtccagaaaaagatgggaaacctgtttagctcacccgtgagcccatagttaaaactctttagacaa NO: 310 caggttgtttccgtttacagagaacaataatattgggtggtgagcatctgtgtgggggttggggtgggataggggatacggggagagt ggagaaaaaggggacacagggttaatgtgaagtccaggatccccctctacatttaaagttggtttaagttggctttaattaatagcaact cttaagataatcagaattttcttaaccttttagccttactgttgaaaagccctgtgatcttgtacaaatcatttgcttcttggatagtaatt tcttttactaaaatgtgggcttttgactagatgaatgtaaatgttcttctagctctgatatcctttattctttatattttctaacagattctg tgtagtgggatgagcagagaacaaaaacaaaataatccagtgagaaaagcccgtaaataaaccttcagaccagagatctattctctagcttat tttaagctcaacttaaaaagaagaactgttctctgattcttttcgccttcaatacacttaatgatttaactccaccctccttcaaaagaaaca gcatttcctacttttatactgtctatatgattgatttgcacagctcatctggccagaagagctgagacatccgttcccctacaagaaactctc cccggtaagtaacctctcagctgcttggcctgttagttagcttctgagatgagtaaaagactttacaggaaacccatagaagacatttggca aacaccaagtgctcatacaattatcttaaaatataatctttaagataaggaaagggtcacagtttggaatgagtttcagacggttataacat caaagatacaaaacatgattgtgagtgaaagactttaaagggagcaatagtattttaataactaacaatccttacctctcaaaagaaagatt tgcagagagatgagtcttagctgaaatcttgaaatcttatcttctgctaaggagaactaaaccctctccagtgagatgccttctgaatatgt gcccacaagaagttgtgtctaagtctggttctcttttttctttttcctccagacaagagggaagcctaaaaatggtcaaaattaatattaaa ttacaaacgccaaataaaattttcctctaatatatcagtttcatggcacagttagtatataattctttatggttcaaaattaaaaatgagct tttctaggggcttctctcagctgcctagtctaaggtgcagggagtttgagactcacagggtttaataagagaaaattctcagctagagcagct gaacttaaatagactaggcaagacagctggttataagactaaactacccagaatgcatgacattcatctgtggtggcagacgaaacatttttt attatattatttcttgggtatgtatgacaactcttaattgtggcaactcagaaactacaaacacaaacttcacagaaaatgtgaggattttac aattggctgttgtcatctatgaccttccctgggacttgggcacccggccatttcactctgactacatcatgtcaccaaacatctgatggtctt gccttttaattctcttttcgaggactgagagggagggtagcatggtagttaagagtgcaggcttcccgcattcaaaatcggttgcttactagct gtgtggctttgagcaagttactcaccctctctgtgcttcaaggtccttgtctgcaaaatgtgaaaaatatttcctgcctcataaggttgccct aaggattaaatgaatgaatgggtatgatgcttagaacagtgattggcatccagtatgtgccctcgaggcctcttaattattactggcttgct catagtgcatgttctttgtgggctaactctagcgtcaataaaaatgttaagactgagttgcagccgggcatggtggctcatgcctgtaatc ccagcattctaggaggctgaggcaggaggatcgcttgagcccaggagttcgagaccagcctgggcaacatagtgtgatcttgtatct ataaaaataaacaaaattagcttggtgtggtggcgcctgtagtccccagccacttggaggggtgaggtgagaggattgcttgagcccg ggatggtccaggctgcagtgagccatgatcgtgccactgcactccagcctgggcgacagagtgagaccctgtctcacaacaacaac aacaacaacaaaaaggctgagctgcaccatgcttgacccagtttcttaaaattgttgtcaaagcttcattcactccatggtgctatagagc acaagattttatttggtgagatggtgctttcatgaattcccccaacagagccaagctctccatctagtggacagggaagctagcagcaa accttcccttcactacaaaacttcattgcttggccaaaaagagagttaattcaatgtagacatctatgtaggcaattaaaaacctattgatgt ataaaacagtttgcattcatggagggcaactaaatacattctaggactttataaaagatcactttttatttatgcacagggtggaacaagat ggattatcaagtgtcaagtccaatctatgacatcaattattatacatcggagccctgccaaaaaatcaatgtgaagcaaatcgcagcccg cctcctgcctccgctctactcactggtgttcatctttggttttgtgggcaacatgctggtcatcctcatcctgataaactgcaaaaggctga agagcatgactgacatctacctgctcaacctggccatctctgacctgtttttccttcttactgtccccttctgggctcactatgctgccgccc agtgggactttggaaatacaatgtgtcaactcttgacagggctctattttataggcttcttctctggaatcttcttcatcatcctcctgacaat cgataggtacctggctgtcgtccatgctgtgtttgctttaaaagccaggacggtcacctttggggtggtgacaagtgtgatcacttgggt ggtggctgtgtttgcgtctctcccaggaatcatctttaccagatctcaaaaagaaggtcttcattacacctgcagctctcattttccatacag tcagtatcaattctggaagaatttccagacattaaagatagtcatcttggggctggtcctgccgctgcttgtcatggtcatctgctactcgg gaatcctaaaaactctgcttcggtgtcgaaatgagaagaagaggcacagggctgtgaggcttatcttcaccatcatgattgtttattttctc ttctgggctccctacaacattgtccttctcctgaacaccttccaggaattctttggcctgaataattgcagtagctctaacaggttggacca agctatgcaggtgacagagactcttgggatgacgcactgctgcatcaaccccatcatctatgcctttgtcggggagaagttcagaaact acctcttagtcttcttccaaaagcacattgccaaacgcttctgcaaatgctgttctattttccagcaagaggctcccgagcgagcaagctc agtttacacccgatccactggggagcaggaaatatctgtgggcttgtgacacggactcaagtgggctggtgacccagtcagagttgtg cacatggcttagttttcatacacagcctgggctgggggtggggtgggagaggtcttttttaaaaggaagttactgttatagagggtctaa gattcatccatttatttggcatctgtttaaagtagattagatcttttaagcccatcaattatagaaagccaaatcaaaatatgttgatgaaaaat agcaacctttttatctccccttcacatgcatcaagttattgacaaactctcccttcactccgaaagttccttatgtatatttaaaagaaagcct cagagaattgctgattcttgagtttagtgatctgaacagaaataccaaaattatttcagaaatgtacaactttttacctagtacaaggcaac atataggttgtaaatgtgtttaaaacaggtctttgtcttgctatggggagaaaagacatgaatatgattagtaaagaaatgacacttttcatg tgtgatttcccctccaaggtatggttaataagtttcactgacttagaaccaggcgagagacttgtggcctgggagagctggggaagctt cttaaatgagaaggaatttgagttggatcatctattgctggcaaagacagaagcctcactgcaagcactgcatgggcaagcttggctgt agaaggagacagagctggttgggaagacatggggaggaaggacaaggctagatcatgaagaaccttgacggcattgctccgtcta agtcatgagctgagcagggagatcctggttggtgttgcagaaggtttactctgtggccaaaggagggtcaggaaggatgagcatttag ggcaaggagaccaccaacagccctcaggtcagggtgaggatggcctctgctaagctcaaggcgtgaggatgggaaggagggag gtattcgtaaggatgggaaggagggaggtattcgtgcagcatatgaggatgcagagtcagcagaactggggtggatttgggttggaa gtgagggtcagagaggagtcagagagaatccctagtcttcaagcagattggagaaacccttgaaaagacatcaagcacagaagga ggaggaggaggtttaggtcaagaagaagatggattggtgtaaaaggatgggtctggtttgcagagcttgaacacagtctcacccaga ctccaggctgtctttcactgaatgcttctgacttcatagatttccttcccatcccagctgaaatactgaggggtctccaggaggagactag atttatgaatacacgaggtatgaggtctaggaacatacttcagctcacacatgagatctaggtgaggattgattacctagtagtcatttcat gggttgttgggaggattctatgaggcaaccacaggcagcatttagcacatactacacattcaataagcatcaaactcttagttactcattc agggatagcactgagcaaagcattgagcaaaggggtcccatagaggtgagggaagcctgaaaaactaagatgctgcctgcccagt gcacacaagtgtaggtatcattttctgcatttaaccgtcaataggcaaaggggggaagggacatattcatttggaaataagctgccttga gccttaaaacccacaaaagtacaatttaccagcctccgtatttcagactgaatgggggtggggggggcgccttaggtacttattccaga tgccttctccagacaaaccagaagcaacagaaaaaatcgtctctccctccctttgaaatgaatataccccttagtgtttgggtatattcattt caaagggagagagagaggtttttttctgttctgtctcatatgattgtgcacatacttgagactgttttgaatttgggggatggctaaaaccat catagtacaggtaaggtgagggaatagtaagtggtgagaactactcagggaatgaaggtgtcagaataataagaggtgctactgactt tctcagcctctgaatatgaacggtgagcattgtggctgtcagcaggaagcaacgaagggaaatgtctttccttttgctcttaagttgtgga gagtgcaacagtagcataggaccctaccctctgggccaagtcaaagacattctgacatcttagtatttgcatattcttatgtatgtgaaagt tacaaattgcttgaaagaaaatatgcatctaataaaaaacaccttctaaaataattcattatattcttgctctttcagtcaagtgtacatttaga gaatagcacataaaactgccagagcattttataagcagctgttttcttccttagtgtgtgtgcatgtgtgtgtgatgtatacaaagagagag ataattgtatttttgtattttcttttaaataatttttaaaattgacccttttcctgagacaaattgccagaatagtttgtatttagagatggt acctctaagagtaaggttgctggttgctgagcaattgacttgaaaacttttaaaattcaaattttaattccactactcaaaagaattgccatg ttttaaaaaagagaattggtgccataagttagttgtctatgtttgaaaatgaagaagatatgcaacgtcatggcctggtcacttacccgcagc cctgagttgtaggcacatcatatgtgagaatgaggatgcttttctttcatttaaaatccctccccaaaacttggctctaattgcagtcatga caatcatgtacatttggatttatgtgcacgagtctcttaccctgagagaggacaggtgctacaggtggaggggacccgtctgggtcacgttcac at

Mutated gene sequences (variants detected after genome sequencing, showing coding sequence only) TP53 > P223fs SEQ ID atggaggagccgcagtcagatcctagcgtcgagcc NO: 311 ccctctgagtcaggaaacattttcagacctatgga aactacttcctgaaaacaacgttctgtcccccttg ccgtcccaagcaatggatgatttgatgctgtcccc ggacgatattgaacaatggttcactgaagacccag gtccagatgaagctcccagaatgccagaggctgct ccccccgtggcccctgcaccagcagctcctacacc ggcggcccctgcaccagccccctcctggcccctgt catcttctgtcccttcccagaaaacctaccagggc agctacggtttccgtctgggcttcttgcattctgg gacagccaagtctgtgacttgcacgtactcccctg ccctcaacaagatgttttgccaactggccaagacc tgccctgtgcagctgtgggttgattccacaccccc gcccggcacccgcgtccgcgccatggccatctaca agcagtcacagcacatgacggaggttgtgaggcgc tgcccccaccatgagcgctgctcagatagcgatgg tctggcccctcctcagcatcttatccgagtggaag gaaatttgcgtgtggagtatttggatgacagaaac acttttcgacatagtgtggtggtgccctatgagcc gcgaggttggctctgactgtaccaccatccactac aactacatgtgtaacagttcctgcatgggcggcat gaaccggaggcccatcctcaccatcatcacactgg aagactccagtggtaatctactgggacggaacagc tttgaggtgcgtgtttgtgcctgtcctgggagaga ccggcgcacagaggaagagaatctccgcaagaaag gggagcctcaccacgagctgcccccagggagcact aagcgagcactgcccaacaacaccagctcctctcc ccagccaaagaagaaaccactggatggagaatatt tcacccttcagatccgtgggcgtgagcgcttcgag atgttccgagagctgaatgaggccttggaactcaa ggatgcccaggctgggaaggagccaggggggagca gggctcactccagccacctgaagtccaaaaagggt cagtctacctcccgccataaaaaactcatgttcaa gacagaagggcctgactcagactga TP53 > 222_224del SEQ ID atggaggagccgcagtcagatcctagcgtcgagcc NO: 312 ccctctgagtcaggaaacattttcagacctatgga aactacttcctgaaaacaacgttctgtcccccttg ccgtcccaagcaatggatgatttgatgctgtcccc ggacgatattgaacaatggttcactgaagacccag gtccagatgaagctcccagaatgccagaggctgct ccccccgtggcccctgcaccagcagctcctacacc ggcggcccctgcaccagccccctcctggcccctgt catcttctgtcccttcccagaaaacctaccagggc agctacggtttccgtctgggcttcttgcattctgg gacagccaagtctgtgacttgcacgtactcccctg ccctcaacaagatgttttgccaactggccaagacc tgccctgtgcagctgtgggttgattccacaccccc gcccggcacccgcgtccgcgccatggccatctaca agcagtcacagcacatgacggaggttgtgaggcgc tgcccccaccatgagcgctgctcagatagcgatgg tctggcccctcctcagcatcttatccgagtggaag gaaatttgcgtgtggagtatttggatgacagaaac acttttcgacatagtgtggtggtgccctatgaggt tggctctgactgtaccaccatccactacaactaca tgtgtaacagttcctgcatgggcggcatgaaccgg aggcccatcctcaccatcatcacactggaagactc cagtggtaatctactgggacggaacagctttgagg tgcgtgtttgtgcctgtcctgggagagaccggcgc acagaggaagagaatctccgcaagaaaggggagcc tcaccacgagctgcccccagggagcactaagcgag cactgcccaacaacaccagctcctctccccagcca aagaagaaaccactggatggagaatatttcaccct tcagatccgtgggcgtgagcgcttcgagatgttcc gagagctgaatgaggccttggaactcaaggatgcc caggctgggaaggagccaggggggagcagggctca ctccagccacctgaagtccaaaaagggtcagtcta cctcccgccataaaaaactcatgttcaagacagaa gggcctgactcagactga CDKN2A > R46fs SEQ ID atggagccggcggcggggagcagcatggagccttc NO: 313 ggctgactggctggccacggccgcggcccggggtc gggtagaggaggtgcgggcgctgctggaggcgggg gcgctgcccaacgcaccgaatagttacggtcgagg ccgatccaggtcatgatgatgggcagcgcccgagt ggcggagctgctgctgctccacggcgcggagccca actgcgccgaccccgccactctcacccgacccgtg cacgacgctgcccgggagggcttcctggacacgct ggtggtgctgcaccgggccggggcgcggctggacg tgcgcgatgcctggggccgtctgcccgtggacctg gctgaggagctgggccatcgcgatgtcgcacggta cctgcgcgcggctgcggggggcaccagaggcagta accatgcccgcatagatgccgcggaaggtccctca gacatccccgattga CDKN2A > 44 45del SEQ ID atggagccggcggcggggagcagcatggagccttc NO: 314 ggctgactggctggccacggccgcggcccggggtc gggtagaggaggtgcgggcgctgctggaggcgggg gcgctgcccaacgcaccgaatagtcggaggccgat ccaggtcatgatgatgggcagcgcccgagtggcgg agctgctgctgctccacggcgcggagcccaactgc gccgaccccgccactctcacccgacccgtgcacga cgctgcccgggagggcttcctggacacgctggtgg tgctgcaccgggccggggcgcggctggacgtgcgc gatgcctggggccgtctgcccgtggacctggctga ggagctgggccatcgcgatgtcgcacggtacctgc gcgcggctgcggggggcaccagaggcagtaaccat gcccgcatagatgccgcggaaggtccctcagacat ccccgattga NOTCH1 > C292fs SEQ ID atgccgccgctcctggcgcccctgctctgcctggc NO: 315 gctgctgcccgcgctcgccgcacgaggcccgcgat gctcccagcccggtgagacctgcctgaatggcggg aagtgtgaagcggccaatggcacggaggcctgcgt ctgtggcggggccttcgtgggcccgcgatgccagg accccaacccgtgcctcagcaccccctgcaagaac gccgggacatgccacgtggtggaccgcagaggcgt ggcagactatgcctgcagctgtgccctgggcttct ctgggcccctctgcctgacacccctggacaatgcc tgcctcaccaacccctgccgcaacgggggcacctg cgacctgctcacgctgacggagtacaagtgccgct gcccgcccggctggtcagggaaatcgtgccagcag gctgacccgtgcgcctccaacccctgcgccaacgg tggccagtgcctgcccttcgaggcctcctacatct gccactgcccacccagcttccatggccccacctgc cggcaggatgtcaacgagtgtggccagaagcccgg gctttgccgccacggaggcacctgccacaacgagg tcggctcctaccgctgcgtctgccgcgccacccac actggccccaactgcgagcggccctacgtgccctg cagcccctcgccctgccagaacgggggcacctgcc gccccacgggcgacgtcacccacgagtgtgcctgc ctgccaggcttcaccggccagaactgtgaggaaaa tatcgacgattgtccaggaaacaactgcaagaacg ggggtgcctgtgtggacggcgtgaacacctacaac tgccgctgcccgccagagtggacaggtcagtacta ccgaggatgtggacgagtgccagctgatgccaaat gcctgccagaacggcgggacctgccacaacaccca cggtggctacaactgcgtgtgtgtcaacggctgga ctggtgaggactgcagcgagaacattgatgactgt gccagcgccgcctgcttccacggcgccacctgcca tgaccgtgtggcctccttctactgcgagtgtcccc atggccgcacaggtctgctgtgccacctcaacgac gcatgcatcagcaacccctgtaacgagggctccaa ctgcgacaccaaccctgtcaatggcaaggccatct gcacctgcccctcggggtacacgggcccggcctgc agccaggacgtggatgagtgctcgctgggtgccaa cccctgcgagcatgcgggcaagtgcatcaacacgc tgggctccttcgagtgccagtgtctgcagggctac acgggcccccgatgcgagatcgacgtcaacgagtg cgtctcgaacccgtgccagaacgacgccacctgcc tggaccagattggggagttccagtgcatctgcatg cccggctacgagggtgtgcactgcgaggtcaacac agacgagtgtgccagcagcccctgcctgcacaatg gccgctgcctggacaagatcaatgagttccagtgc gagtgccccacgggcttcactgggcatctgtgcca gtacgatgtggacgagtgtgccagcaccccctgca agaatggtgccaagtgcctggacggacccaacact tacacctgtgtgtgcacggaagggtacacggggac gcactgcgaggtggacatcgatgagtgcgaccccg acccctgccactacggctcctgcaaggacggcgtc gccaccttcacctgcctctgccgcccaggctacac gggccaccactgcgagaccaacatcaacgagtgct ccagccagccctgccgccacgggggcacctgccag gaccgcgacaacgcctacctctgcttctgcctgaa ggggaccacaggacccaactgcgagatcaacctgg atgactgtgccagcagcccctgcgactcgggcacc tgtctggacaagatcgatggctacgagtgtgcctg tgagccgggctacacagggagcatgtgtaacatca acatcgatgagtgtgcgggcaacccctgccacaac gggggcacctgcgaggacggcatcaatggcttcac ctgccgctgccccgagggctaccacgaccccacct gcctgtctgaggtcaatgagtgcaacagcaacccc tgcgtccacggggcctgccgggacagcctcaacgg gtacaagtgcgactgtgaccctgggtggagtggga ccaactgtgacatcaacaacaatgagtgtgaatcc aacccttgtgtcaacggcggcacctgcaaagacat gaccagtggctacgtgtgcacctgccgggagggct tcagcggtcccaactgccagaccaacatcaacgag tgtgcgtccaacccatgtctgaaccagggcacgtg tattgacgacgttgccgggtacaagtgcaactgcc tgctgccctacacaggtgccacgtgtgaggtggtg ctggccccgtgtgcccccagcccctgcagaaacgg cggggagtgcaggcaatccgaggactatgagagct tctcctgtgtctgccccacgggctggcaagggcag acctgtgaggtcgacatcaacgagtgcgttctgag cccgtgccggcacggcgcatcctgccagaacaccc acggcggctaccgctgccactgccaggccggctac agtgggcgcaactgcgagaccgacatcgacgactg ccggcccaacccgtgtcacaacgggggctcctgca cagacggcatcaacacggccttctgcgactgcctg cccggcttccggggcactttctgtgaggaggacat caacgagtgtgccagtgacccctgccgcaacgggg ccaactgcacggactgcgtggacagctacacgtgc acctgccccgcaggcttcagcgggatccactgtga gaacaacacgcctgactgcacagagagctcctgct tcaacggtggcacctgcgtggacggcatcaactcg ttcacctgcctgtgtccacccggcttcacgggcag ctactgccagcacgatgtcaatgagtgcgactcac agccctgcctgcatggcggcacctgtcaggacggc tgcggctcctacaggtgcacctgcccccagggcta cactggccccaactgccagaaccttgtgcactggt gtgactcctcgccctgcaagaacggcggcaaatgc tggcagacccacacccagtaccgctgcgagtgccc cagcggctggaccggcctttactgcgacgtgccca gcgtgtcctgtgaggtggctgcgcagcgacaaggt gttgacgttgcccgcctgtgccagcatggagggct ctgtgtggacgcgggcaacacgcaccactgccgct gccaggcgggctacacaggcagctactgtgaggac ctggtggacgagtgctcacccagcccctgccagaa cggggccacctgcacggactacctgggcggctact cctgcaagtgcgtggccggctaccacggggtgaac tgctctgaggagatcgacgagtgcctctcccaccc ctgccagaacgggggcacctgcctcgacctcccca acacctacaagtgctcctgcccacggggcactcag ggtgtgcactgtgagatcaacgtggacgactgcaa tccccccgttgaccccgtgtcccggagccccaagt gctttaacaacggcacctgcgtggaccaggtgggc ggctacagctgcacctgcccgccgggcttcgtggg tgagcgctgtgagggggatgtcaacgagtgcctgt ccaatccctgcgacgcccgtggcacccagaactgc gtgcagcgcgtcaatgacttccactgcgagtgccg tgctggtcacaccgggcgccgctgcgagtccgtca tcaatggctgcaaaggcaagccctgcaagaatggg ggcacctgcgccgtggcctccaacaccgcccgcgg gttcatctgcaagtgccctgcgggcttcgagggcg ccacgtgtgagaatgacgctcgtacctgcggcagc ctgcgctgcctcaacggcggcacatgcatctccgg cccgcgcagccccacctgcctgtgcctgggcccct tcacgggccccgaatgccagttcccggccagcagc ccctgcctgggcggcaacccctgctacaaccaggg gacctgtgagcccacatccgagagccccttctacc gttgcctgtgccccgccaaattcaacgggctcttg tgccacatcctggactacagcttcgggggtggggc cgggcgcgacatccccccgccgctgatcgaggagg cgtgcgagctgcccgagtgccaggaggacgcgggc aacaaggtctgcagcctgcagtgcaacaaccacgc gtgcggctgggacggcggtgactgctccctcaact tcaatgacccctggaagaactgcacgcagtctctg cagtgctggaagtacttcagtgacggccactgtga cagccagtgcaactcagccggctgcctcttcgacg gctttgactgccagcgtgcggaaggccagtgcaac cccctgtacgaccagtactgcaaggaccacttcag cgacgggcactgcgaccagggctgcaacagcgcgg agtgcgagtgggacgggctggactgtgcggagcat gtacccgagaggctggcggccggcacgctggtggt ggtggtgctgatgccgccggagcagctgcgcaaca gctccttccacttcctgcgggagctcagccgcgtg ctgcacaccaacgtggtcttcaagcgtgacgcaca cggccagcagatgatcttcccctactacggccgcg aggaggagctgcgcaagcaccccatcaagcgtgcc gccgagggctgggccgcacctgacgccctgctggg ccaggtgaaggcctcgctgctccctggtggcagcg agggtgggcggcggcggagggagctggaccccatg gacgtccgcggctccatcgtctacctggagattga caaccggcagtgtgtgcaggcctcctcgcagtgct tccagagtgccaccgacgtggccgcattcctggga gcgctcgcctcgctgggcagcctcaacatccccta caagatcgaggccgtgcagagtgagaccgtggagc cgcccccgccggcgcagctgcacttcatgtacgtg gcggcggccgcctttgtgcttctgttcttcgtggg ctgcggggtgctgctgtcccgcaagcgccggcggc agcatggccagctctggttccctgagggcttcaaa gtgtctgaggccagcaagaagaagcggcgggagcc cctcggcgaggactccgtgggcctcaagcccctga agaacgcttcagacggtgccctcatggacgacaac cagaatgagtggggggacgaggacctggagaccaa gaagttccggttcgaggagcccgtggttctgcctg acctggacgaccagacagaccaccggcagtggact cagcagcacctggatgccgctgacctgcgcatgtc tgccatggcccccacaccgccccagggtgaggttg acgccgactgcatggacgtcaatgtccgcgggcct gatggcttcaccccgctcatgatcgcctcctgcag cgggggcggcctggagacgggcaacagcgaggaag aggaggacgcgccggccgtcatctccgacttcatc taccagggcgccagcctgcacaaccagacagaccg cacgggcgagaccgccttgcacctggccgcccgct actcacgctctgatgccgccaagcgcctgctggag gccagcgcagatgccaacatccaggacaacatggg ccgcaccccgctgcatgcggctgtgtctgccgacg cacaaggtgtcttccagatcctgatccggaaccga gccacagacctggatgcccgcatgcatgatggcac gacgccactgatcctggctgcccgcctggccgtgg agggcatgctggaggacctcatcaactcacacgcc gacgtcaacgccgtagatgacctgggcaagtccgc cctgcactgggccgccgccgtgaacaatgtggatg ccgcagttgtgctcctgaagaacggggctaacaaa gatatgcagaacaacagggaggagacacccctgtt tctggccgcccgggagggcagctacgagaccgcca aggtgctgctggaccactttgccaaccgggacatc acggatcatatggaccgcctgccgcgcgacatcgc acaggagcgcatgcatcacgacatcgtgaggctgc tggacgagtacaacctggtgcgcagcccgcagctg cacggagccccgctggggggcacgcccaccctgtc gcccccgctctgctcgcccaacggctacctgggca gcctcaagcccggcgtgcagggcaagaaggtccgc aagcccagcagcaaaggcctggcctgtggaagcaa ggaggccaaggacctcaaggcacggaggaagaagt cccaggacggcaagggctgcctgctggacagctcc ggcatgctctcgcccgtggactccctggagtcacc ccatggctacctgtcagacgtggcctcgccgccac tgctgccctccccgttccagcagtctccgtccgtg cccctcaaccacctgcctgggatgcccgacaccca cctgggcatcgggcacctgaacgtggcggccaagc ccgagatggcggcgctgggtgggggcggccggctg gcctttgagactggcccacctcgtctctcccacct gcctgtggcctctggcaccagcaccgtcctgggct ccagcagcggaggggccctgaatttcactgtgggc gggtccaccagtttgaatggtcaatgcgagtggct gtcccggctgcagagcggcatggtgccgaaccaat acaaccctctgcgggggagtgtggcaccaggcccc ctgagcacacaggccccctccctgcagcatggcat ggtaggcccgctgcacagtagccttgctgccagcg ccctgtcccagatgatgagctaccagggcctgccc agcacccggctggccacccagcctcacctggtgca gacccagcaggtgcagccacaaaacttacagatgc agcagcagaacctgcagccagcaaacatccagcag cagcaaagcctgcagccgccaccaccaccaccaca gccgcaccttggcgtgagctcagcagccagcggcc acctgggccggagcttcctgagtggagagccgagc caggcagacgtgcagccactgggccccagcagcct ggcggtgcacactattctgccccaggagagccccg ccctgcccacgtcgctgccatcctcgctggtccca cccgtgaccgcagcccagttcctgacgcccccctc gcagcacagctactcctcgcctgtggacaacaccc ccagccaccagctacaggtgcctgagcaccccttc ctcaccccgtcccctgagtcccctgaccagtggtc cagctcgtccccgcattccaacgtctccgactggt ccgagggcgtctccagccctcccaccagcatgcag tcccagatcgcccgcattccggaggccttcaagta a NOTCHI > T293fs SEQ ID atgccgccgctcctggcgcccctgctctgcctggc NO: 316 gctgctgcccgcgctcgccgcacgaggcccgcgat gctcccagcccggtgagacctgcctgaatggcggg aagtgtgaagcggccaatggcacggaggcctgcgt ctgtggcggggccttcgtgggcccgcgatgccagg accccaacccgtgcctcagcaccccctgcaagaac gccgggacatgccacgtggtggaccgcagaggcgt ggcagactatgcctgcagctgtgccctgggcttct ctgggcccctctgcctgacacccctggacaatgcc tgcctcaccaacccctgccgcaacgggggcacctg cgacctgctcacgctgacggagtacaagtgccgct gcccgcccggctggtcagggaaatcgtgccagcag gctgacccgtgcgcctccaacccctgcgccaacgg tggccagtgcctgcccttcgaggcctcctacatct gccactgcccacccagcttccatggccccacctgc cggcaggatgtcaacgagtgtggccagaagcccgg gctttgccgccacggaggcacctgccacaacgagg tcggctcctaccgctgcgtctgccgcgccacccac actggccccaactgcgagcggccctacgtgccctg cagcccctcgccctgccagaacgggggcacctgcc gccccacgggcgacgtcacccacgagtgtgcctgc ctgccaggcttcaccggccagaactgtgaggaaaa tatcgacgattgtccaggaaacaactgcaagaacg ggggtgcctgtgtggacggcgtgaacacctacaac tgccgctgcccgccagagtggacaggtcagtactg tactcgaggatgtggacgagtgccagctgatgcca aatgcctgccagaacggcgggacctgccacaacac ccacggtggctacaactgcgtgtgtgtcaacggct ggactggtgaggactgcagcgagaacattgatgac tgtgccagcgccgcctgcttccacggcgccacctg ccatgaccgtgtggcctccttctactgcgagtgtc cccatggccgcacaggtctgctgtgccacctcaac gacgcatgcatcagcaacccctgtaacgagggctc caactgcgacaccaaccctgtcaatggcaaggcca tctgcacctgcccctcggggtacacgggcccggcc tgcagccaggacgtggatgagtgctcgctgggtgc caacccctgcgagcatgcgggcaagtgcatcaaca cgctgggctccttcgagtgccagtgtctgcagggc tacacgggcccccgatgcgagatcgacgtcaacga gtgcgtctcgaacccgtgccagaacgacgccacct gcctggaccagattggggagttccagtgcatctgc atgcccggctacgagggtgtgcactgcgaggtcaa cacagacgagtgtgccagcagcccctgcctgcaca atggccgctgcctggacaagatcaatgagttccag tgcgagtgccccacgggcttcactgggcatctgtg ccagtacgatgtggacgagtgtgccagcaccccct gcaagaatggtgccaagtgcctggacggacccaac acttacacctgtgtgtgcacggaagggtacacggg gacgcactgcgaggtggacatcgatgagtgcgacc ccgacccctgccactacggctcctgcaaggacggc gtcgccaccttcacctgcctctgccgcccaggcta cacgggccaccactgcgagaccaacatcaacgagt gctccagccagccctgccgccacgggggcacctgc caggaccgcgacaacgcctacctctgcttctgcct gaaggggaccacaggacccaactgcgagatcaacc tggatgactgtgccagcagcccctgcgactcgggc acctgtctggacaagatcgatggctacgagtgtgc ctgtgagccgggctacacagggagcatgtgtaaca tcaacatcgatgagtgtgcgggcaacccctgccac aacgggggcacctgcgaggacggcatcaatggctt cacctgccgctgccccgagggctaccacgacccca cctgcctgtctgaggtcaatgagtgcaacagcaac ccctgcgtccacggggcctgccgggacagcctcaa cgggtacaagtgcgactgtgaccctgggtggagtg ggaccaactgtgacatcaacaacaatgagtgtgaa tccaacccttgtgtcaacggcggcacctgcaaaga catgaccagtggctacgtgtgcacctgccgggagg gcttcagcggtcccaactgccagaccaacatcaac gagtgtgcgtccaacccatgtctgaaccagggcac gtgtattgacgacgttgccgggtacaagtgcaact gcctgctgccctacacaggtgccacgtgtgaggtg gtgctggccccgtgtgcccccagcccctgcagaaa cggcggggagtgcaggcaatccgaggactatgaga gcttctcctgtgtctgccccacgggctggcaaggg cagacctgtgaggtcgacatcaacgagtgcgttct gagcccgtgccggcacggcgcatcctgccagaaca cccacggcggctaccgctgccactgccaggccggc tacagtgggcgcaactgcgagaccgacatcgacga ctgccggcccaacccgtgtcacaacgggggctcct gcacagacggcatcaacacggccttctgcgactgc ctgcccggcttccggggcactttctgtgaggagga catcaacgagtgtgccagtgacccctgccgcaacg gggccaactgcacggactgcgtggacagctacacg tgcacctgccccgcaggcttcagcgggatccactg tgagaacaacacgcctgactgcacagagagctcct gcttcaacggtggcacctgcgtggacggcatcaac tcgttcacctgcctgtgtccacccggcttcacggg cagctactgccagcacgatgtcaatgagtgcgact cacagccctgcctgcatggcggcacctgtcaggac ggctgcggctcctacaggtgcacctgcccccaggg ctacactggccccaactgccagaaccttgtgcact ggtgtgactcctcgccctgcaagaacggcggcaaa tgctggcagacccacacccagtaccgctgcgagtg ccccagcggctggaccggcctttactgcgacgtgc ccagcgtgtcctgtgaggtggctgcgcagcgacaa ggtgttgacgttgcccgcctgtgccagcatggagg gctctgtgtggacgcgggcaacacgcaccactgcc gctgccaggcgggctacacaggcagctactgtgag gacctggtggacgagtgctcacccagcccctgcca gaacggggccacctgcacggactacctgggcggct actcctgcaagtgcgtggccggctaccacggggtg aactgctctgaggagatcgacgagtgcctctccca cccctgccagaacgggggcacctgcctcgacctcc ccaacacctacaagtgctcctgcccacggggcact cagggtgtgcactgtgagatcaacgtggacgactg caatccccccgttgaccccgtgtcccggagcccca agtgctttaacaacggcacctgcgtggaccaggtg ggcggctacagctgcacctgcccgccgggcttcgt gggtgagcgctgtgagggggatgtcaacgagtgcc tgtccaatccctgcgacgcccgtggcacccagaac tgcgtgcagcgcgtcaatgacttccactgcgagtg ccgtgctggtcacaccgggcgccgctgcgagtccg tcatcaatggctgcaaaggcaagccctgcaagaat gggggcacctgcgccgtggcctccaacaccgcccg cgggttcatctgcaagtgccctgcgggcttcgagg gcgccacgtgtgagaatgacgctcgtacctgcggc agcctgcgctgcctcaacggcggcacatgcatctc cggcccgcgcagccccacctgcctgtgcctgggcc ccttcacgggccccgaatgccagttcccggccagc agcccctgcctgggcggcaacccctgctacaacca ggggacctgtgagcccacatccgagagccccttct accgttgcctgtgccccgccaaattcaacgggctc ttgtgccacatcctggactacagcttcgggggtgg ggccgggcgcgacatccccccgccgctgatcgagg aggcgtgcgagctgcccgagtgccaggaggacgcg ggcaacaaggtctgcagcctgcagtgcaacaacca cgcgtgcggctgggacggcggtgactgctccctca acttcaatgacccctggaagaactgcacgcagtct ctgcagtgctggaagtacttcagtgacggccactg tgacagccagtgcaactcagccggctgcctcttcg acggctttgactgccagcgtgcggaaggccagtgc aaccccctgtacgaccagtactgcaaggaccactt cagcgacgggcactgcgaccagggctgcaacagcg cggagtgcgagtgggacgggctggactgtgcggag catgtacccgagaggctggcggccggcacgctggt ggtggtggtgctgatgccgccggagcagctgcgca acagctccttccacttcctgcgggagctcagccgc gtgctgcacaccaacgtggtcttcaagcgtgacgc acacggccagcagatgatcttcccctactacggcc gcgaggaggagctgcgcaagcaccccatcaagcgt gccgccgagggctgggccgcacctgacgccctgct gggccaggtgaaggcctcgctgctccctggtggca gcgagggtgggcggcggcggagggagctggacccc atggacgtccgcggctccatcgtctacctggagat tgacaaccggcagtgtgtgcaggcctcctcgcagt gcttccagagtgccaccgacgtggccgcattcctg ggagcgctcgcctcgctgggcagcctcaacatccc ctacaagatcgaggccgtgcagagtgagaccgtgg agccgcccccgccggcgcagctgcacttcatgtac gtggcggcggccgcctttgtgcttctgttcttcgt gggctgcggggtgctgctgtcccgcaagcgccggc ggcagcatggccagctctggttccctgagggcttc aaagtgtctgaggccagcaagaagaagcggcggga gcccctcggcgaggactccgtgggcctcaagcccc tgaagaacgcttcagacggtgccctcatggacgac aaccagaatgagtggggggacgaggacctggagac caagaagttccggttcgaggagcccgtggttctgc ctgacctggacgaccagacagaccaccggcagtgg actcagcagcacctggatgccgctgacctgcgcat gtctgccatggcccccacaccgccccagggtgagg ttgacgccgactgcatggacgtcaatgtccgcggg cctgatggcttcaccccgctcatgatcgcctcctg cagcgggggcggcctggagacgggcaacagcgagg aagaggaggacgcgccggccgtcatctccgacttc atctaccagggcgccagcctgcacaaccagacaga ccgcacgggcgagaccgccttgcacctggccgccc gctactcacgctctgatgccgccaagcgcctgctg gaggccagcgcagatgccaacatccaggacaacat gggccgcaccccgctgcatgcggctgtgtctgccg acgcacaaggtgtcttccagatcctgatccggaac cgagccacagacctggatgcccgcatgcatgatgg cacgacgccactgatcctggctgcccgcctggccg tggagggcatgctggaggacctcatcaactcacac gccgacgtcaacgccgtagatgacctgggcaagtc cgccctgcactgggccgccgccgtgaacaatgtgg atgccgcagttgtgctcctgaagaacggggctaac aaagatatgcagaacaacagggaggagacacccct gtttctggccgcccgggagggcagctacgagaccg ccaaggtgctgctggaccactttgccaaccgggac atcacggatcatatggaccgcctgccgcgcgacat cgcacaggagcgcatgcatcacgacatcgtgaggc tgctggacgagtacaacctggtgcgcagcccgcag ctgcacggagccccgctggggggcacgcccaccct gtcgcccccgctctgctcgcccaacggctacctgg gcagcctcaagcccggcgtgcagggcaagaaggtc cgcaagcccagcagcaaaggcctggcctgtggaag caaggaggccaaggacctcaaggcacggaggaaga agtcccaggacggcaagggctgcctgctggacagc tccggcatgctctcgcccgtggactccctggagtc accccatggctacctgtcagacgtggcctcgccgc cactgctgccctccccgttccagcagtctccgtcc gtgcccctcaaccacctgcctgggatgcccgacac ccacctgggcatcgggcacctgaacgtggcggcca agcccgagatggcggcgctgggtgggggcggccgg ctggcctttgagactggcccacctcgtctctccca cctgcctgtggcctctggcaccagcaccgtcctgg gctccagcagcggaggggccctgaatttcactgtg ggcgggtccaccagtttgaatggtcaatgcgagtg gctgtcccggctgcagagcggcatggtgccgaacc aatacaaccctctgcgggggagtgtggcaccaggc cccctgagcacacaggccccctccctgcagcatgg catggtaggcccgctgcacagtagccttgctgcca gcgccctgtcccagatgatgagctaccagggcctg cccagcacccggctggccacccagcctcacctggt gcagacccagcaggtgcagccacaaaacttacaga tgcagcagcagaacctgcagccagcaaacatccag cagcagcaaagcctgcagccgccaccaccaccacc acagccgcaccttggcgtgagctcagcagccagcg gccacctgggccggagcttcctgagtggagagccg agccaggcagacgtgcagccactgggccccagcag cctggcggtgcacactattctgccccaggagagcc ccgccctgcccacgtcgctgccatcctcgctggtc ccacccgtgaccgcagcccagttcctgacgccccc ctcgcagcacagctactcctcgcctgtggacaaca cccccagccaccagctacaggtgcctgagcacccc ttcctcaccccgtcccctgagtcccctgaccagtg gtccagctcgtccccgcattccaacgtctccgact ggtccgagggcgtctccagccctcccaccagcatg cagtcccagatcgcccgcattccggaggccttcaa gtaa

Representative coding sequences for targeting: Tp53 SEQ ID atggaggagccgcagtcagatcctagcgtcgagccccctctgagtcagga NO: 324 aacattttcagacctatggaaactacttcctgaaaacaacgttctgtccc ccttgccgtcccaagcaatggatgatttgatgctgtccccggacgatatt gaacaatggttcactgaagacccaggtccagatgaagctcccagaatgcc agaggctgctccccccgtggcccctgcaccagcagctcctacaccggcgg cccctgcaccagccccctcctggcccctgtcatcttctgtcccttcccag aaaacctaccagggcagctacggtttccgtctgggcttcttgcattctgg gacagccaagtctgtgacttgcacgtactcccctgccctcaacaagatgt tttgccaactggccaagacctgccctgtgcagctgtgggttgattccaca cccccgcccggcacccgcgtccgcgccatggccatctacaagcagtcaca gcacatgacggaggttgtgaggcgctgcccccaccatgagcgctgctcag atagcgatggtctggcccctcctcagcatcttatccgagtggaaggaaat ttgcgtgtggagtatttggatgacagaaacacttttcgacatagtgtggt ggtgccctatgagccgcctgaggttggctctgactgtaccaccatccact acaactacatgtgtaacagttcctgcatgggcggcatgaaccggaggccc atcctcaccatcatcacactggaagactccagtggtaatctactgggacg gaacagctttgaggtgcgtgtttgtgcctgtcctgggagagaccggcgca cagaggaagagaatctccgcaagaaaggggagcctcaccacgagctgccc ccagggagcactaagcgagcactgcccaacaacaccagctcctctcccca gccaaagaagaaaccactggatggagaatatttcacccttcaggaccaga ccagctttcaaaaagaaaattgttaa CDKN2A SEQ ID atggtgcgcaggttcttggtgaccctccggattcggcgcgcgtgcggccc NO: 325 gccgcgagtgagggttttcgtggttcacatcccgcggctcacgggggagt gggcagcgccaggggcgcccgccgctgtggccctcgtgctgatgctactg aggagccagcgtctagggcagcagccgcttcctagaagaccaggtcatga tgatgggcagcgcccgagtggcggagctgctgctgctccacggcgcggag cccaactgcgccgaccccgccactctcacccgacccgtgcacgacgctgc ccgggagggcttcctggacacgctggtggtgctgcaccgggccggggcgc ggctggacgtgcgcgatgcctggggccgtctgcccgtggacctggctga NOTCH1 SEQ ID atgccgccgctcctggcgcccctgctctgcctggcgctgctgcccgcgct NO: 326 cgccgcacgaggcccgcgatgctcccagcccggtgagacctgcctgaatg gcgggaagtgtgaagcggccaatggcacggaggcctgcgtctgtggcggg gccttcgtgggcccgcgatgccaggaccccaacccgtgcctcagcacccc ctgcaagaacgccgggacatgccacgtggtggaccgcagaggcgtggcag actatgcctgcagctgtgccctgggcttctctgggcccctctgcctgaca cccctggacaatgcctgcctcaccaacccctgccgcaacgggggcacctg cgacctgctcacgctgacggagtacaagtgccgctgcccgcccggctggt cagggaaatcgtgccagcaggctgacccgtgcgcctccaacccctgcgcc aacggtggccagtgcctgcccttcgaggcctcctacatctgccactgccc acccagcttccatggccccacctgccggcaggatgtcaacgagtgtggcc agaagcccgggctttgccgccacggaggcacctgccacaacgaggtcggc tcctaccgctgcgtctgccgcgccacccacactggccccaactgcgagcg gccctacgtgccctgcagcccctcgccctgccagaacgggggcacctgcc gccccacgggcgacgtcacccacgagtgtgcctgcctgccaggcttcacc ggccagaactgtgaggaaaatatcgacgattgtccaggaaacaactgcaa gaacgggggtgcctgtgtggacggcgtgaacacctacaactgccgctgcc cgccagagtggacaggtcagtactgtaccgaggatgtggacgagtgccag ctgatgccaaatgcctgccagaacggcgggacctgccacaacacccacgg tggctacaactgcgtgtgtgtcaacggctggactggtgaggactgcagcg agaacattgatgactgtgccagcgccgcctgcttccacggcgccacctgc catgaccgtgtggcctccttctactgcgagtgtccccatggccgcacagg tctgctgtgccacctcaacgacgcatgcatcagcaacccctgtaacgagg gctccaactgcgacaccaaccctgtcaatggcaaggccatctgcacctgc ccctcggggtacacgggcccggcctgcagccaggacgtggatgagtgctc gctgggtgccaacccctgcgagcatgcgggcaagtgcatcaacacgctgg gctccttcgagtgccagtgtctgcagggctacacgggcccccgatgcgag atcgacgtcaacgagtgcgtctcgaacccgtgccagaacgacgccacctg cctggaccagattggggagttccagtgcatctgcatgcccggctacgagg gtgtgcactgcgaggtcaacacagacgagtgtgccagcagcccctgcctg cacaatggccgctgcctggacaagatcaatgagttccagtgcgagtgccc cacgggcttcactgggcatctgtgccagtacgatgtggacgagtgtgcca gcaccccctgcaagaatggtgccaagtgcctggacggacccaacacttac acctgtgtgtgcacggaagggtacacggggacgcactgcgaggtggacat cgatgagtgcgaccccgacccctgccactacggctcctgcaaggacggcg tcgccaccttcacctgcctctgccgcccaggctacacgggccaccactgc gagaccaacatcaacgagtgctccagccagccctgccgccacgggggcac ctgccaggaccgcgacaacgcctacctctgcttctgcctgaaggggacca caggacccaactgcgagatcaacctggatgactgtgccagcagcccctgc gactcgggcacctgtctggacaagatcgatggctacgagtgtgcctgtga gccgggctacacagggagcatgtgtaacatcaacatcgatgagtgtgcgg gcaacccctgccacaacgggggcacctgcgaggacggcatcaatggcttc acctgccgctgccccgagggctaccacgaccccacctgcctgtctgaggt caatgagtgcaacagcaacccctgcgtccacggggcctgccgggacagcc tcaacgggtacaagtgcgactgtgaccctgggtggagtgggaccaactgt gacatcaacaacaatgagtgtgaatccaacccttgtgtcaacggcggcac ctgcaaagacatgaccagtggctacgtgtgcacctgccgggagggcttca gcggtcccaactgccagaccaacatcaacgagtgtgcgtccaacccatgt ctgaaccagggcacgtgtattgacgacgttgccgggtacaagtgcaactg cctgctgccctacacaggtgccacgtgtgaggtggtgctggccccgtgtg cccccagcccctgcagaaacggcggggagtgcaggcaatccgaggactat gagagcttctcctgtgtctgccccacgggctggcaagggcagacctgtga ggtcgacatcaacgagtgcgttctgagcccgtgccggcacggcgcatcct gccagaacacccacggcggctaccgctgccactgccaggccggctacagt gggcgcaactgcgagaccgacatcgacgactgccggcccaacccgtgtca caacgggggctcctgcacagacggcatcaacacggccttctgcgactgcc tgcccggcttccggggcactttctgtgaggaggacatcaacgagtgtgcc agtgacccctgccgcaacggggccaactgcacggactgcgtggacagcta cacgtgcacctgccccgcaggcttcagcgggatccactgtgagaacaaca cgcctgactgcacagagagctcctgcttcaacggtggcacctgcgtggac ggcatcaactcgttcacctgcctgtgtccacccggcttcacgggcagcta ctgccagcacgatgtcaatgagtgcgactcacagccctgcctgcatggcg gcacctgtcaggacggctgcggctcctacaggtgcacctgcccccagggc tacactggccccaactgccagaaccttgtgcactggtgtgactcctcgcc ctgcaagaacggcggcaaatgctggcagacccacacccagtaccgctgcg agtgccccagcggctggaccggcctttactgcgacgtgcccagcgtgtcc tgtgaggtggctgcgcagcgacaaggtgttgacgttgcccgcctgtgcca gcatggagggctctgtgtggacgcgggcaacacgcaccactgccgctgcc aggcgggctacacaggcagctactgtgaggacctggtggacgagtgctca cccagcccctgccagaacggggccacctgcacggactacctgggcggcta ctcctgcaagtgcgtggccggctaccacggggtgaactgctctgaggaga tcgacgagtgcctctcccacccctgccagaacgggggcacctgcctcgac ctccccaacacctacaagtgctcctgcccacggggcactcagggtgtgca ctgtgagatcaacgtggacgactgcaatccccccgttgaccccgtgtccc ggagccccaagtgctttaacaacggcacctgcgtggaccaggtgggcggc tacagctgcacctgcccgccgggcttcgtgggtgagcgctgtgaggggga tgtcaacgagtgcctgtccaatccctgcgacgcccgtggcacccagaact gcgtgcagcgcgtcaatgacttccactgcgagtgccgtgctggtcacacc gggcgccgctgcgagtccgtcatcaatggctgcaaaggcaagccctgcaa gaatgggggcacctgcgccgtggcctccaacaccgcccgcgggttcatct gcaagtgccctgcgggcttcgagggcgccacgtgtgagaatgacgctcgt acctgcggcagcctgcgctgcctcaacggcggcacatgcatctccggccc gcgcagccccacctgcctgtgcctgggccccttcacgggccccgaatgcc agttcccggccagcagcccctgcctgggcggcaacccctgctacaaccag gggacctgtgagcccacatccgagagccccttctaccgttgcctgtgccc cgccaaattcaacgggctcttgtgccacatcctggactacagcttcgggg gtggggccgggcgcgacatccccccgccgctgatcgaggaggcgtgcgag ctgcccgagtgccaggaggacgcgggcaacaaggtctgcagcctgcagtg caacaaccacgcgtgcggctgggacggcggtgactgctccctcaacttca atgacccctggaagaactgcacgcagtctctgcagtgctggaagtacttc agtgacggccactgtgacagccagtgcaactcagccggctgcctcttcga cggctttgactgccagcgtgcggaaggccagtgcaaccccctgtacgacc agtactgcaaggaccacttcagcgacgggcactgcgaccagggctgcaac agcgcggagtgcgagtgggacgggctggactgtgcggagcatgtacccga gaggctggcggccggcacgctggtggtggtggtgctgatgccgccggagc agctgcgcaacagctccttccacttcctgcgggagctcagccgcgtgctg cacaccaacgtggtcttcaagcgtgacgcacacggccagcagatgatctt cccctactacggccgcgaggaggagctgcgcaagcaccccatcaagcgtg ccgccgagggctgggccgcacctgacgccctgctgggccaggtgaaggcc tcgctgctccctggtggcagcgagggtgggcggcggcggagggagctgga ccccatggacgtccgcggctccatcgtctacctggagattgacaaccggc agtgtgtgcaggcctcctcgcagtgcttccagagtgccaccgacgtggcc gcattcctgggagcgctcgcctcgctgggcagcctcaacatcccctacaa gatcgaggccgtgcagagtgagaccgtggagccgcccccgccggcgcagc tgcacttcatgtacgtggcggcggccgcctttgtgcttctgttcttcgtg ggctgcggggtgctgctgtcccgcaagcgccggcggcagcatggccagct ctggttccctgagggcttcaaagtgtctgaggccagcaagaagaagcggc gggagcccctcggcgaggactccgtgggcctcaagcccctgaagaacgct tcagacggtgccctcatggacgacaaccagaatgagtggggggacgagga cctggagaccaagaagttccggttcgaggagcccgtggttctgcctgacc tggacgaccagacagaccaccggcagtggactcagcagcacctggatgcc gctgacctgcgcatgtctgccatggcccccacaccgccccagggtgaggt tgacgccgactgcatggacgtcaatgtccgcgggcctgatggcttcaccc cgctcatgatcgcctcctgcagcgggggcggcctggagacgggcaacagc gaggaagaggaggacgcgccggccgtcatctccgacttcatctaccaggg cgccagcctgcacaaccagacagaccgcacgggcgagaccgccttgcacc tggccgcccgctactcacgctctgatgccgccaagcgcctgctggaggcc agcgcagatgccaacatccaggacaacatgggccgcaccccgctgcatgc ggctgtgtctgccgacgcacaaggtgtcttccagatcctgatccggaacc gagccacagacctggatgcccgcatgcatgatggcacgacgccactgatc ctggctgcccgcctggccgtggagggcatgctggaggacctcatcaactc acacgccgacgtcaacgccgtagatgacctgggcaagtccgccctgcact gggccgccgccgtgaacaatgtggatgccgcagttgtgctcctgaagaac ggggctaacaaagatatgcagaacaacagggaggagacacccctgtttct ggccgcccgggagggcagctacgagaccgccaaggtgctgctggaccact ttgccaaccgggacatcacggatcatatggaccgcctgccgcgcgacatc gcacaggagcgcatgcatcacgacatcgtgaggctgctggacgagtacaa cctggtgcgcagcccgcagctgcacggagccccgctggggggcacgccca ccctgtcgcccccgctctgctcgcccaacggctacctgggcagcctcaag cccggcgtgcagggcaagaaggtccgcaagcccagcagcaaaggcctggc ctgtggaagcaaggaggccaaggacctcaaggcacggaggaagaagtccc aggacggcaagggctgcctgctggacagctccggcatgctctcgcccgtg gactccctggagtcaccccatggctacctgtcagacgtggcctcgccgcc actgctgccctccccgttccagcagtctccgtccgtgcccctcaaccacc tgcctgggatgcccgacacccacctgggcatcgggcacctgaacgtggcg gccaagcccgagatggcggcgctgggtgggggcggccggctggcctttga gactggcccacctcgtctctcccacctgcctgtggcctctggcaccagca ccgtcctgggctccagcagcggaggggccctgaatttcactgtgggcggg tccaccagtttgaatggtcaatgcgagtggctgtcccggctgcagagcgg catggtgccgaaccaatacaaccctctgcgggggagtgtggcaccaggcc ccctgagcacacaggccccctccctgcagcatggcatggtaggcccgctg cacagtagccttgctgccagcgccctgtcccagatgatgagctaccaggg cctgcccagcacccggctggccacccagcctcacctggtgcagacccagc aggtgcagccacaaaacttacagatgcagcagcagaacctgcagccagca aacatccagcagcagcaaagcctgcagccgccaccaccaccaccacagcc gcaccttggcgtgagctcagcagccagcggccacctgggccggagcttcc tgagtggagagccgagccaggcagacgtgcagccactgggccccagcagc ctggcggtgcacactattctgccccaggagagccccgccctgcccacgtc gctgccatcctcgctggtcccacccgtgaccgcagcccagttcctgacgc ccccctcgcagcacagctactcctcgcctgtggacaacacccccagccac cagctacaggtgcctgagcaccccttcctcaccccgtcccctgagtcccc tgaccagtggtccagctcgtccccgcattccaacgtctccgactggtccg agggcgtctccagccctcccaccagcatgcagtcccagatcgcccgcatt ccggaggccttcaagtaa FATI SEQ ID atggggagacatttggctttgctcctgcttctgctccttctcttccaaca NO: 327 ttttggagacagtgatggcagccaacgacttgaacagactcctctgcagt ttacacacctcgagtacaacgtcaccgtgcaggagaactctgcagctaag acttatgtggggcatcctgtcaagatgggtgtttacattacacatccagc gtgggaagtaaggtacaaaattgtttccggagacagtgaaaacctgttca aagctgaagagtacattctcggagacttttgctttctaagaataaggacc aaaggaggaaatacagctattcttaatagagaagtgaaggatcactacac attgatagtgaaagcacttgaaaaaaatactaatgtggaggcgcgaacaa aggtcagggtgcaggtgctggatacaaatgacttgagaccgttattctca cccacctcatacagcgtttctttacctgaaaacacagctataaggaccag tatcgcaagagtcagcgccacggatgcagacataggaaccaacggggaat tttactacagttttaaagatcgaacagatatgtttgctattcacccaacc agtggtgtgatagtgttaactggtagacttgattacctagagaccaagct ctatgagatggaaatcctcgctgcggaccgtggcatgaagttgtatggga gcagtggcatcagcagcatggccaagctaacggtgcacatcgaacaggcc aatgaatgtgctccggtgataacagcagtgacattgtcaccatcagaact ggacagggacccagcatatgcaattgtgacagtggatgactgcgatcagg gtgccaatggtgacatagcatctttaagcatcgtggcaggtgaccttctc cagcagtttagaacagtgaggtcctttccagggagtaaggagtataaagt caaagccatcggtggcattgattgggacagtcatcctttcggctacaatc tcacactacaggctaaagataaaggaactccgccccagttctcttctgtt aaagtcattcacgtgacttctccacagttcaaagccgggccagtcaagtt tgaaaaggatgtttacagagcagaaataagtgaatttgctcctcccaaca cacctgtggtcatggtaaaggccattcctgcttattcccatttgaggtat gtttttaaaagtacacctggaaaagctaaattcagtttaaattacaacac tggtctcatttctattttagaaccagttaaaagacagcaggcagcccatt ttgaacttgaagtaacaacaagtgacagaaaagcgtccaccaaggtcttg gtgaaagtcttaggtgcaaatagcaatccccctgaatttacccagacagc gtacaaagctgcttttgatgagaacgtgcccattggtactactgtcatga gcctgagtgccgtagaccctgatgagggtgagaacgggtacgtgacatac agtatcgcaaatttaaatcatgtgccgtttgcgattgaccatttcactgg tgccgtgagtacgtcagaaaacctggactacgaactgatgcctcgggttt atactctgaggattcgtgcatcagactggggcttgccgtaccgccgggaa gtcgaagtccttgctacaattactctcaataacttgaatgacaacacacc tttgtttgagaaaataaattgtgaagggacaattcccagagatctaggcg tgggagagcaaataaccactgtttctgctattgatgcagatgaacttcag ttggtacagtatcagattgaagctggaaatgaactggatttctttagttt aaaccccaactcgggggtattgtcattaaagcgatcgctaatggatggct taggtgcaaaggtgtctttccacagtctgagaatcacagctacagatgga gaaaattttgccacaccattatatatcaacataacagtggctgccagtca caagctggtaaacttgcagtgtgaagagactggtgttgccaaaatgctgg cagagaagctcctgcaggcaaataaattacacaaccagggagaggtggag gatattttcttcgattctcactctgtcaatgctcacataccgcagtttag aagcactcttccgactggtattcaggtaaaggaaaaccagcctgtgggtt ccagtgtaattttcatgaactccactgaccttgacactggcttcaatgga aaactggtctatgctgtttctggaggaaatgaggatagttgcttcatgat tgatatggaaacaggaatgctgaaaattttatctcctcttgaccgtgaaa caacagacaaatacaccctgaatattaccgtctatgaccttgggataccc cagaaggctgcgtggcgtcttctacatgtcgtggttgtcgatgccaatga taatccacccgagtttttacaggagagctattttgtggaagtgagtgaag acaaggaggtacatagtgaaatcatccaggttgaagccacagataaagac ctggggcccaacggacacgtgacgtactcaattgttacagacacagacac attttcaattgacagcgtgacgggtgttgttaacatcgcacgccctctgg atcgagagctgcagcatgagcactccttaaagattgaggccagggaccaa gccagagaagagcctcagctgttctccactgtcgttgtgaaagtatcact agaagatgttaatgacaacccacctacatttattccacctaattatcgtg tgaaagtccgagaggatcttccagaaggaaccgtcatcatgtggttagaa gcccacgatcctgatttaggtcagtctggtcaggtgagatacagccttct ggaccacggagaaggaaacttcgatgtggataaactcagtggagcagtta ggatcgtccagcagttggactttgagaagaagcaagtgtataatctcact gtgagggccaaagacaagggaaagccagtttctctgtcttctacttgcta tgttgaagttgaggtggttgatgtgaatgagaacctgcacccacccgtgt tttccagctttgtggaaaaggggacagtgaaagaagatgcacctgttggt tcattggtaatgacggtgtcggctcatgatgaggacgccagaagagatgg ggagatccgatactccattagagatggctctggcgttggtgttttcaaaa taggtgaagagacaggtgtcatagagacgtcagatcgactggaccgtgaa tcgacctcccattattggctaacagtctttgcaaccgatcagggtgtcgt gcctctttcatcgttcatagagatctacatagaggttgaggatgtcaatg acaatgcaccacagacatcagagcctgtttattacccagaaatcatggaa aattctcctaaagatgtatctgtggtccagatcgaggcatttgatccaga ttcgagctctaatgacaagctcatgtacaaaattacaagtggaaatccac aaggattcttttcaatacatcctaaaacaggtctcatcacaactacgtca aggaagctagaccgagaacagcaagatgaacacatattagaggttactgt gacagacaatggtagtccccccaaatcaaccattgcaagagtcattgtga aaatccttgatgaaaatgacaacaaacctcagtttctgcaaaagttctac aaaatcagactccctgagcgggaaaagccagaccgagaaagaaatgccag acgggagccgctctatcacgtcatagccaccgacaaggatgagggcccca atgcagaaatctcctacagcatcgaagacgggaatgagcatggcaaattt ttcatcgaaccgaaaactggagtggtttcgtccaagaggttttcagcagc tggagaatatgatattctttcaattaaggcagttgacaatggtcgccctc aaaagtcatcaaccaccagactccatattgaatggatctccaagcccaaa ccgtccctggagcccatttcatttgaagaatcattttttacctttactgt gatggaaagtgaccccgttgctcacatgattggagtaatatctgtggagc ctcctggcatacccctttggtttgacatcactggtggcaactacgacagt cacttcgatgtggacaagggaactggaaccatcattgttgccaaacctct tgatgcagaacagaagtcaaactacaacctcacagtcgaggctacagatg gaaccaccactatcctcactcaggtattcatcaaagtaatagacacaaat gaccatcgtcctcagttttctacatcaaagtatgaagttgttattcctga agatacagcgccagaaacagaaattttgcaaatcagtgctgtggatcagg atgagaaaaacaaactaatctacactctgcagagcagtagagatccactg agtctcaagaaatttcgtcttgatcctgcaaccggctctctctatacttc tgagaaactggatcatgaagctgttcaccagcacaccctcacggtcatgg tacgagatcaagatgtgcctgtaaaacgcaactttgcaaggattgtggtc aatgtcagcgacacgaatgaccacgccccgtggttcaccgcttcctccta caaagggcgggtttatgaatcggcagccgttggctcagttgtgttgcagg tgacggctctggacaaggacaaagggaaaaatgctgaagtgctgtactcg atcgagtcaggaaatattggaaattcttttatgattgatcctgtcttggg ctctattaaaactgccaaagaattagatcgaagtaaccaagcggagtatg atttaatggtaaaagctacagataagggcagtccaccaatgagtgaaata acttctgtgcgtatctttgtcacaattgctgacaacgcctctccgaagtt tacatcaaaagaatattctgttgaacttagtgaaactgtcagcattggga gtttcgttgggatggttacagcccatagtcaatcatcagtggtgtatgaa ataaaagatggaaatacaggtgatgcttttgatattaatccacattctgg aactatcatcactcagaaagccctggactttgaaactttgcccatttaca cattgataatacaaggaactaacatggctggtttgtccactaatacaacg gttctagttcacttgcaggatgagaatgacaacgcgccagtttttatgca ggcagaatatacaggactcattagtgaatcagcctcaattaacagcgtgg tcctaacagacaggaatgtcccactggtgattcgagcagctgatgctgat aaagactcaaatgctttgcttgtatatcacattgttgaaccatctgtaca cacatattttgctattgattctagcactggtgctattcatacagtactaa gtctggactatgaagaaacaagtatttttcactttaccgtccaagtgcat gacatgggaaccccacgtttatttgctgagtatgcagcgaatgtaacagt acatgtaattgacattaatgactgcccccctgtgtttgccaagccattat atgaagcatctcttttgttaccaacatacaaaggagtaaaagtcatcaca gtaaatgctacagatgctgattcaagtgcattctcacagttgatttactc catcaccgaaggcaacatcggggagaagttttctatggactacaagactg gtgctctcactgtccaaaacacaactcagttaagaagccgctacgagcta accgttagagcttccgatggcagatttgccggccttacctctgtcaaaat taatgtgaaagaaagcaaagaaagtcacctaaagtttacccaggatgtct actctgcggtagtgaaagagaattccaccgaggccgaaacattagctgtc attactgctattgggaatccaatcaatgagcctttgttttatcacatcct caacccagatcgcagatttaaaataagccgcacttcaggagttctgtcaa ccactggcacgcccttcgatcgtgagcagcaggaggcgtttgatgtggtt gtagaagtgacagaggaacataagccttctgcagtggcccacgttgtcgt gaaggtcattgtagaagaccaaaatgataatgcgccggtgtttgtcaacc ttccctactacgccgttgttaaagtggacactgaggtgggccatgtcatt cgctatgtcactgctgtagacagagacagtggcagaaacggggaagtgca ttactacctcaaggaacatcatgaacactttcaaattggacccttgggtg aaatttcactgaaaaagcaatttgagcttgacaccttaaataaagaatat cttgttacagtggttgcaaaagatggagggaacccggccttttcagcgga agttatcgttccgatcactgtcatgaataaagccatgcctgtgtttgaaa aacctttctacagtgcagagattgcagagagcatccaggtgcacagccct gtggtccacgtgcaggctaacagcccggaaggcctgaaagtgttctacag catcacagacggagaccctttcagccagttcactattaacttcaatactg gagttatcaatgtcatagctcctctggactttgaggcccacccggcatat aagctgagcatacgcgcaactgactccttgacgggcgctcatgctgaagt atttgtggacatcatagtagacgacatcaatgataaccctcctgtgtttg ctcagcagtcttatgcggtgaccctgtctgaggcatctgtaattggaacg tctgttgttcaagttagagccaccgattctgattcagaaccaaatagagg aatctcataccagatgtttgggaatcacagcaagagtcatgatcattttc atgtagacagcagcactggcctcatctcactactcagaaccctggattac gagcagtcccggcagcacacgatttttgtgagggcagttgatggtggtat gcccacgctgagcagtgatgtgattgtcacggtggacgttaccgacctca atgataatccaccactctttgaacaacagatttatgaagccagaattagc gagcacgcccctcatgggcatttcgtgacctgtgtaaaagcctatgatgc agacagttcagacatagacaagttgcagtattccattctgtctggcaatg atcataaacattttgtcattgacagtgcaacagggattatcaccctctca aacctgcaccggcacgccctgaagccattttacagtcttaacctgtcagt gtctgatggagtttttagaagttccacccaggttcatgtaactgtaattg gaggcaatttgcacagtcctgctttccttcagaacgaatatgaagtggaa ctagctgaaaacgctcccctacataccctggtgatggaggtgaaaactac ggatggggattctggtatttatggtcacgttacttaccatattgtaaatg actttgccaaagacagattttacataaatgagagaggacagatatttact ttggaaaaacttgatcgagaaaccccggcggagaaagtgatctcagtccg tttaatggctaaggatgctggaggaaaagttgctttctgcaccgtgaatg tcatccttacagatgacaatgacaatgcaccacaatttcgagcaaccaaa tacgaagtgaatatcgggtccagtgctgctaaagggacttcagtcgttaa agttcttgcaagtgatgccgatgagggctccaatgccgacatcacctatg ccattgaagcagactctgaaagtgtaaaagagaatttggaaattaacaaa ctgtccggcgtaatcactacaaaggagagcctcattggcttggaaaatga attcttcactttctttgttagagctgtggataatgggtctccatcaaaag aatctgttgttcttgtctatgttaaaatccttccaccggaaatgcagctt ccaaaattttcagaacctttctatacctttacagtgtcagaggacgtgcc tattggaacagagatagatctcatccgagcagaacatagtgggactgttc tttacagcctggtcaaagggaatactccagaaagcaatagggatgagtcc tttgtgattgacagacagagcgggagactgaagttggagaagagtcttga tcatgagacaactaagtggtatcagttttccatactggccaggtgcactc aagatgaccatgagatggtggcttctgtagatgttagtatccaagtgaaa gatgcaaatgacaacagcccggtctttgaatctagtccatatgaggcatt cattgttgaaaacctgccagggggaagtagagtaattcagatcagggcat ctgatgctgactcaggaaccaacggccaagttatgtatagcctggatcag tcacaaagtgtggaagtcattgaatcctttgccattaacatggaaacagg ctggattacaactttaaaggaacttgaccatgaaaagagagacaattacc agattaaagtggttgcatcagatcatggtgaaaagatccagctatcctcc acagccattgtggatgttaccgtcaccgatgtcaacgatagtccaccacg attcacggccgagatctataaagggactgtgagtgaggatgacccccaag gtggggtgattgccatcttaagtaccacggatgctgattctgaagagatc aacagacaagttacatatttcataacaggaggggatcctttaggacagtt tgccgttgaaactatacagaatgaatggaaggtatatgtgaagaaacctc tagacagggaaaaaagggacaattaccttcttactatcacggcaactgat ggcaccttctcatcaaaagcgatagttgaagtgaaagttctggatgcaaa tgacaacagtccagtttgtgaaaagactttatattcagacactattcctg aagacgtccttcctggaaaattgatcatgcagatctctgctacagacgca gacatccgctctaacgctgaaattacttacacgttattgggttcaggtgc agaaaaattcaaactaaatccagacacaggtgaactgaaaacgtcaaccc cccttgatcgtgaggagcaagctgtttatcatcttctcgtcagggccaca gatggaggaggaagattctgccaagccagtattgtgctcacgctagaaga tgtgaacgataacgcccccgaattctctgccgatccttatgccatcaccg tgtttgaaaacacagagccgggaacgctgctgacaagagtgcaggccaca gatgccgacgcaggattaaatcggaagattttatactcactgattgactc tgctgatgggcagttctccattaacgaattatctggaattattcagttag aaaaacctttggacagagaactccaggcagtatacaccctctctttgaaa gctgtggatcaaggcttgccaaggaggctgactgccactggcactgtgat tgtatcagttcttgacataaatgacaacccccctgtgtttgagtaccgtg aatatggtgccaccgtgtctgaggacattcttgttggaactgaagttctt caagtgtatgcagcaagtcgggatattgaagcaaatgcagaaatcaccta ctcaataataagtggaaatgaacatgggaaattcagcatagattctaaaa caggggccgtatttatcattgagaatctggattatgagagctctcatgag tattacctaacagtagaggccactgatggaggcacgccttcactgagcga cgttgccactgtgaacgttaatgtaacagatatcaacgataatacccctg tgttcagccaagacacctacacgacagtcatcagtgaagatgccgttctt gagcagtctgtcatcacggttatggccgatgatgccgatggaccttccaa cagccacatccactactcaattatagatggcaaccaaggaagctcgttca caattgaccccgtcaggggagaagtcaaagtgaccaaacttctcgaccga gaaacgatttcaggttacacgctcacggttcaagcttctgataatggcag tccacccagagtcaacacgacgaccgtgaacatcgatgtgtccgatgtca atgacaacgcgcccgtcttctccaggggaaactacagtgtcattatccag gaaaataagccagtgggcttcagcgtgctgcagctggtagtaacagatga ggattcttcccataacggtccacccttcttctttactattgtaactggaa atgatgagaaggcttttgaagttaacccgcaaggagtcctcctgacatca tctgccatcaagaggaaggagaaagatcattacttactgcaggtgaaggt ggcagataatggaaagcctcagttgtcatctttgacatacattgacatta gggtaattgaggagagcatctatccgcctgcgattttgcccctggagatt ttcatcacctcttctggagaagaatactcaggtggcgtcattgggaagat ccatgccacagaccaggacgtgtatgatactctaacctacagtctcgacc ctcagatggacaacctgttctctgtttccagcacagggggcaagctgata gcacacaaaaagctagacatagggcaataccttctcaatgtcagcgtaac agatgggaagttcacgacggtggccgacatcacagtgcatatcagacaag tcacacaggagatgttgaaccacaccatcgcgatccgctttgccaacctc actccggaagaattcgttggtgactactggcgcaacttccagcgagcttt acggaacatcctgggtgtgaggaggaacgacatacagattgttagtttgc agtcctctgaacctcacccacatctggacgtcttactttttgtagagaaa ccaggtagtgctcagatctcaacaaaacaacttctgcacaagattaactc ttccgtgactgacattgaggaaatcattggagttaggatactgaatgtat tccagaaactctgcgcgggactggactgcccctggaagttctgcgatgaa aaggtgtctgtggatgaaagtgtgatgtcaacacacagcacagccagact gagttttgtgactccccgccaccacagggcagcggtgtgtctctgcaaag agggaaggtgcccacctgtccaccatggctgtgaagatgatccgtgccct gagggatccgaatgtgtgtctgatccctgggaggagaaacacacctgtgt ctgtcccagcggcaggtttggtcagtgcccagggagttcatctatgacac tgactggaaacagctacgtgaaataccgtctgacggaaaatgaaaacaaa ttagagatgaaactgaccatgaggctcagaacatattccacgcatgcggt tgtcatgtatgctcgaggaactgactatagcatcttggagattcatcatg gaaggctgcagtacaagtttgactgtggaagtggccctggaattgtctct gttcagagcattcaggtcaatgatgggcagtggcacgcagtggccctgga agtgaatggaaactatgctcgcttggttctagaccaagttcatactgcat cgggcacagccccagggactctgaaaaccctgaacctggataactatgtg ttttttggtggccacatccgtcagcagggaacaaggcatggaagaagtcc tcaagttggtaatggtttcaggggttgtatggactccatttatttgaatg ggcaggagctccctttaaacagcaaacccagaagctatgcacacatcgaa gagtcggtggatgtatctccaggctgcttcctgacggccacggaagactg cgccagcaacccttgccagaatggaggcgtttgcaatccgtcacctgctg gaggttattactgcaaatgcagtgccttgtacatagggacccactgtgag ataagcgtcaatccgtgttcctccaagccatgcctctatgggggcacgtg tgttgtcgacaacggaggctttgtttgccagtgtagaggattatatactg gtcagaggtgtcagcttagtccatactgcaaagatgaaccctgtaagaat ggcggaacatgctttgacagtttggatggcgccgtttgtcagtgtgattc gggttttaggggagaaaggtgtcagagtgatatcgacgagtgctctggaa acccttgcctgcacggggccctctgtgagaacacgcacggctcctatcac tgcaactgcagccacgagtacaggggacgtcactgcgaggatgctgcgcc caaccagtatgtgtccacgccgtggaacattgggttggcggaaggaattg gaatcgttgtgtttgttgcagggatatttttactggtggtggtgtttgtt ctctgccgtaagatgattagtcggaaaaagaagcatcaggctgaacctaa agacaagcacctgggacccgctacggctttcttgcaaagaccgtattttg attccaagctaaataagaacatttactcagacataccaccccaggtgcct gtccggcctatttcctacaccccgagtattccaagtgactcaagaaacaa tctggaccgaaattccttcgaaggatctgctatcccagagcatcccgaat tcagcacttttaaccccgagtctgtgcacgggcaccgaaaagcagtggcg gtctgcagcgtggcgccaaacctgcctcccccacccccttcaaactcccc ttctgacagcgactccatccagaagcctagctgggactttgactatgaca caaaagtggtggatcttgatccctgtctttccaagaagcctctagaggaa aagccttcccagccatacagtgcccgggaaagcctgtctgaagtgcagtc tctgagctccttccagtccgaatcgtgcgatgacaatgggtatcactggg atacatcagattggatgccaagcgttcctctgccggacatacaagagttc cccaactatgaggtgattgatgagcagacacccctgtactcagcagatcc aaacgccatcgatacggactattaccctggaggctacgacatcgaaagtg attttcctccacccccagaagacttccccgcagctgatgagctaccaccg ttaccgcccgaattcagcaatcagtttgaatccatccaccctcctagaga catgcctgccgcgggtagcttgggttcttcatcaagaaaccggcagaggt tcaacttgaatcagtatttgcccaatttttatcccctcgatatgtctgaa cctcaaacaaaaggcactggtgagaatagtacttgtagagaaccccatgc cccttacccgccagggtatcaaagacacttcgaggcgcccgctgtcgaga gcatgcccatgtctgtgtacgcctccaccgcctcctgctctgacgtgtca gcctgctgcgaagtggagtccgaggtcatgatgagtgactatgagagcgg ggacgacggccacttcgaagaggtgacgatcccgcccctggattcccagc agcacacggaagtctga RBI SEQ ID atgccgcccaaaaccccccgaaaaacggccgccaccgccgccgctgccgc NO: 328 cgcggaacccccggcaccgccgccgccgccccctcctgaggaggacccag agcaggacagcggcccggaggacctgcctctcgtcaggcttgagtttgaa gaaacagaagaacctgattttactgcattatgtcagaaattaaagatacc agatcatgtcagagagagagcttggttaacttgggagaaagtttcatctg tggatggagtattgggaggttatattcaaaagaaaaaggaactgtgggga atctgtatctttattgcagcagttgacctagatgagatgtcgttcacttt tactgagctacagaaaaacatagaaatcagtgtccataaattctttaact tactaaaagaaattgataccagtaccaaagttgataatgctatgtcaaga ctgttgaagaagtatgatgtattgtttgcactcttcagcaaattggaaag gacatgtgaacttatatatttgacacaacccagcagttcgatatctactg aaataaattctgcattggtgctaaaagtttcttggatcacatttttatta gctaaaggggaagtattacaaatggaagatgatctggtgatttcatttca gttaatgctatgtgtccttgactattttattaaactctcacctcccatgt tgctcaaagaaccatataaaacagctgttatacccattaatggttcacct cgaacacccaggcgaggtcagaacaggagtgcacggatagcaaaacaact agaaaatgatacaagaattattgaagttctctgtaaagaacatgaatgta atatagatgaggtgaaaaatgtttatttcaaaaattttataccttttatg aattctcttggacttgtaacatctaatggacttccagaggttgaaaatct ttctaaacgatacgaagaaatttatcttaaaaataaagatctagatgcaa gattatttttggatcatgataaaactcttcagactgattctatagacagt tttgaaacacagagaacaccacgaaaaagtaaccttgatgaagaggtgaa tgtaattcctccacacactccagttaggactgttatgaacactatccaac aattaatgatgattttaaattcagcaagtgatcaaccttcagaaaatctg atttcctattttaacaactgcacagtgaatccaaaagaaagtatactgaa aagagtgaaggatataggatacatctttaaagagaaatttgctaaagctg tgggacagggttgtgtcgaaattggatcacagcgatacaaacttggagtt cgcttgtattaccgagtaatggaatccatgcttaaatcagaagaagaacg attatccattcaaaattttagcaaacttctgaatgacaacatttttcata tgtctttattggcgtgcgctcttgaggttgtaatggccacatatagcaga agtacatctcagaatcttgattctggaacagatttgtctttcccatggat tctgaatgtgcttaatttaaaagcctttgatttttacaaagtgatcgaaa gttttatcaaagcagaaggcaacttgacaagagaaatgataaaacattta gaacgatgtgaacatcgaatcatggaatcccttgcatggctctcagattc acctttatttgatcttattaaacaatcaaaggaccgagaaggaccaactg atcaccttgaatctgcttgtcctcttaatcttcctctccagaataatcac actgcagcagatatgtatctttctcctgtaagatctccaaagaaaaaagg ttcaactacgcgtgtaaattctactgcaaatgcagagacacaagcaacct cagccttccagacccagaagccattgaaatctacctctctttcactgttt tataaaaaagtgtatcggctagcctatctccggctaaatacactttgtga acgccttctgtctgagcacccagaattagaacatatcatctggacccttt tccagcacaccctgcagaatgagtatgaactcatgagagacaggcatttg gaccaaattatgatgtgttccatgtatggcatatgcaaagtgaagaatat agaccttaaattcaaaatcattgtaacagcatacaaggatcttcctcatg ctgttcaggagacattcaaacgtgttttgatcaaagaagaggagtatgat tctattatagtattctataactcggtcttcatgcagagactgaaaacaaa tattttgcagtatgcttccaccaggccccctaccttgtcaccaatacctc acattcctcgaagcccttacaagtttcctagttcacccttacggattcct ggagggaacatctatatttcacccctgaagagtccatataaaatttcaga aggtctgccaacaccaacaaaaatgactccaagatcaagaatcttagtat caattggtgaatcattcgggacttctgagaagttccagaaaataaatcag atggtatgtaacagcgaccgtgtgctcaaaagaagtgctgaaggaagcaa ccctcctaaaccactgaaaaaactacgctttgatattgaaggatcagatg aagcagatggaagtaaacatctcccaggagagtccaaatttcagcagaaa ctggcagaaatgacttctactcgaacacgaatgcaaaagcagaaaatgaa tgatagcatggatacctcaaacaaggaagagaaatga CASP8 SEQ ID atggagggaggcagaagagccagggtggttattgaaagtaaaagaaactt NO: 329 cttcctgggagcctttcccacccccttccctgctgagcacgtggagttag gcaggttaggggactcggagactgcgatggtgccaggaaagggtggagcg gattatattctcctgccttttaaaaagatggacttcagcagaaatcttta tgatattggggaacaactggacagtgaagatctggcctccctcaagttcc tgagcctggactacattccgcaaaggaagcaagaacccatcaaggatgcc ttgatgttattccagagactccaggaaaagagaatgttggaggaaagcaa tctgtccttcctgaaggagctgctcttccgaattaatagactggatttgc tgattacctacctaaacactagaaaggaggagatggaaagggaacttcag acaccaggcagggctcaaatttctgcctacagggtcatgctctatcagat ttcagaagaagtgagcagatcagaattgaggtcttttaagtttcttttgc aagaggaaatctccaaatgcaaactggatgatgacatgaacctgctggat attttcatagagatggagaagagggtcatcctgggagaaggaaagttgga catcctgaaaagagtctgtgcccaaatcaacaagagcctgctgaagataa tcaacgactatgaagaattcagcaaagagagaagcagcagccttgaagga agtcctgatgaattttcaaatggggaggagttgtgtggggtaatgacaat ctcggactctccaagagaacaggatagtgaatcacagactttggacaaag tttaccaaatgaaaagcaaacctcggggatactgtctgatcatcaacaat cacaattttgcaaaagcacgggagaaagtgcccaaacttcacagcattag ggacaggaatggaacacacttggatgcaggggctttgaccacgacctttg aagagcttcattttgagatcaagccccacgatgactgcacagtagagcaa atctatgagattttgaaaatctaccaactcatggaccacagtaacatgga ctgcttcatctgctgtatcctctcccatggagacaagggcatcatctatg gcactgatggacaggaggcccccatctatgagctgacatctcagttcact ggtttgaagtgcccttcccttgctggaaaacccaaagtgttttttattca ggcttgtcagggggataactaccagaaaggtatacctgttgagactgatt cagaggagcaaccctatttagaaatggatttatcatcacctcaaacgaga tatatcccggatgaggctgactttctgctggggatggccactgtgaataa ctgtgtttcctaccgaaaccctgcagagggaacctggtacatccagtcac tttgccagagcctgagagagcgatgtcctcgaggcgatgatattctcacc atcctgactgaagtgaactatgaagtaagcaacaaggatgacaagaaaaa catggggaaacagatgcctcagcctactttcacactaagaaaaaaacttg tctteecttctgattga PTEN SEQ ID atgacagccatcatcaaagagatcgttagcagaaacaaaaggagatatca NO: 330 agaggatggattcgacttagacttgacctatatttatccaaacattattg ctatgggatttcctgcagaaagacttgaaggcgtatacaggaacaatatt gatgatgtagtaaggtttttggattcaaagcataaaaaccattacaagat atacaatctttgtgctgaaagacattatgacaccgccaaatttaattgca gagttgcacaatatccttttgaagaccataacccaccacagctagaactt atcaaacccttttgtgaagatcttgaccaatggctaagtgaagatgacaa tcatgttgcagcaattcactgtaaagctggaaagggacgaactggtgtaa tgatatgtgcatatttattacatcggggcaaatttttaaaggcacaagag gccctagatttctatggggaagtaaggaccagagacaaaaagggagtaac tattcccagtcagaggcgctatgtgtattattatagctacctgttaaaga atcatctggattatagaccagtggcactgttgtttcacaagatgatgttt gaaactattccaatgttcagtggcggaacttgcaatcctcagtttgtggt ctgccagctaaaggtgaagatatattcctccaattcaggacccacacgac gggaagacaagttcatgtactttgagttccctcagccgttacctgtgtgt ggtgatatcaaagtagagttcttccacaaacagaacaagatgctaaaaaa ggacaaaatgtttcacttttgggtaaatacattcttcataccaggaccag aggaaacctcagaaaaagtagaaaatggaagtctatgtgatcaagaaatc gatagcatttgcagtatagagcgtgcagataatgacaaggaatatctagt acttactttaacaaaaaatgatcttgacaaagcaaataaagacaaagcca accgatacttttctccaaattttaaggtgaagctgtacttcacaaaaaca gtagaggagccgtcaaatccagaggctagcagttcaacttctgtaacacc agatgttagtgacaatgaacctgatcattatagatattctgacaccactg actctgatccagagaatgaaccttttgatgaagatcagcatacacaaatt acaaaagtctga EPHA2 SEQ ID atggagctccaggcagcccgcgcctgcttcgccctgctgtggggctgtgc NO: 331 gctggccgcggccgcggcggcgcagggcaaggaagtggtactgctggact ttgctgcagctggaggggagctcggctggctcacacacccgtatggcaaa gggtgggacctgatgcagaacatcatgaatgacatgccgatctacatgta ctccgtgtgcaacgtgatgtctggcgaccaggacaactggctccgcacca actgggtgtaccgaggagaggctgagcgtatcttcattgagctcaagttt actgtacgtgactgcaacagcttccctggtggcgccagctcctgcaagga gactttcaacctctactatgccgagtcggacctggactacggcaccaact tccagaagcgcctgttcaccaagattgacaccattgcgcccgatgagatc accgtcagcagcgacttcgaggcacgccacgtgaagctgaacgtggagga gcgctccgtggggccgctcacccgcaaaggcttctacctggccttccagg atatcggtgcctgtgtggcgctgctctccgtccgtgtctactacaagaag tgccccgagctgctgcagggcctggcccacttccctgagaccatcgccgg ctctgatgcaccttccctggccactgtggccggcacctgtgtggaccatg ccgtggtgccaccggggggtgaagagccccgtatgcactgtgcagtggat ggcgagtggctggtgcccattgggcagtgcctgtgccaggcaggctacga gaaggtggaggatgcctgccaggcctgctcgcctggattttttaagtttg aggcatctgagagcccctgcttggagtgccctgagcacacgctgccatcc cctgagggtgccacctcctgcgagtgtgaggaaggcttcttccgggcacc tcaggacccagcgtcgatgccttgcacacgacccccctccgccccacact acctcacagccgtgggcatgggtgccaaggtggagctgcgctggacgccc cctcaggacagcgggggccgcgaggacattgtctacagcgtcacctgcga acagtgctggcccgagtctggggaatgcgggccgtgtgaggccagtgtgc gctactcggagcctcctcacggactgacccgcaccagtgtgacagtgagc gacctggagccccacatgaactacaccttcaccgtggaggcccgcaatgg cgtctcaggcctggtaaccagccgcagcttccgtactgccagtgtcagca tcaaccagacagagccccccaaggtgaggctggagggccgcagcaccacc tcgcttagcgtctcctggagcatccccccgccgcagcagagccgagtgtg gaagtacgaggtcacttaccgcaagaagggagactccaacagctacaatg tgcgccgcaccgagggtttctccgtgaccctggacgacctggccccagac accacctacctggtccaggtgcaggcactgacgcaggagggccagggggc cggcagcaaggtgcacgaattccagacgctgtccccggagggatctggca acttggcggtgattggcggcgtggctgtcggtgtggtcctgcttctggtg ctggcaggagttggcttctttatccaccgcaggaggaagaaccagcgtgc ccgccagtccccggaggacgtttacttctccaagtcagaacaactgaagc ccctgaagacatacgtggacccccacacatatgaggaccccaaccaggct gtgttgaagttcactaccgagatccatccatcctgtgtcactcggcagaa ggtgatcggagcaggagagtttggggaggtgtacaagggcatgctgaaga catcctcggggaagaaggaggtgccggtggccatcaagacgctgaaagcc ggctacacagagaagcagcgagtggacttcctcggcgaggccggcatcat gggccagttcagccaccacaacatcatccgcctagagggcgtcatctcca aatacaagcccatgatgatcatcactgagtacatggagaatggggccctg gacaagttccttcgggagaaggatggcgagttcagcgtgctgcagctggt gggcatgctgcggggcatcgcagctggcatgaagtacctggccaacatga actatgtgcaccgtgacctggctgcccgcaacatcctcgtcaacagcaac ctggtctgcaaggtgtctgactttggcctgtcccgcgtgctggaggacga ccccgaggccacctacaccaccagtggcggcaagatccccatccgctgga ccgccccggaggccatttcctaccggaagttcacctctgccagcgacgtg tggagctttggcattgtcatgtgggaggtgatgacctatggcgagcggcc ctactgggagttgtccaaccacgaggtgatgaaagccatcaatgatggct tccggctccccacacccatggactgcccctccgccatctaccagctcatg atgcagtgctggcagcaggagcgtgcccgccgccccaagttcgctgacat cgtcagcatcctggacaagctcattcgtgcccctgactccctcaagaccc tggctgactttgacccccgcgtgtctatccggctccccagcacgagcggc tcggagggggtgcccttccgcacggtgtccgagtggctggagtccatcaa gatgcagcagtatacggagcacttcatggcggccggctacactgccatcg agaaggtggtgcagatgaccaacgacgacatcaagaggattggggtgcgg ctgcccggccaccagaagcgcatcgcctacagcctgctgggactcaagga ccaggtgaacactgtggggatccccatctga TGFBR2 SEQ ID atgggtcgggggctgctcaggggcctgtggccgctgcacatcgtcctgtg NO: 332 gacgcgtatcgccagcacgatcccaccgcacgttcagaagtcggttaata acgacatgatagtcactgacaacaacggtgcagtcaagtttccacaactg tgtaaattttgtgatgtgagattttccacctgtgacaaccagaaatcctg catgagcaactgcagcatcacctccatctgtgagaagccacaggaagtct gtgtggctgtatggagaaagaatgacgagaacataacactagagacagtt tgccatgaccccaagctcccctaccatgactttattctggaagatgctgc ttctccaaagtgcattatgaaggaaaaaaaaaagcctggtgagactttct tcatgtgttcctgtagctctgatgagtgcaatgacaacatcatcttctca gaagaatataacaccagcaatcctgacttgttgctagtcatatttcaagt gacaggcatcagcctcctgccaccactgggagttgccatatctgtcatca tcatcttctactgctaccgcgttaaccggcagcagaagctgagttcaacc tgggaaaccggcaagacgcggaagctcatggagttcagcgagcactgtgc catcatcctggaagatgaccgctctgacatcagctccacgtgtgccaaca acatcaaccacaacacagagctgctgcccattgagctggacaccctggtg gggaaaggtcgctttgctgaggtctataaggccaagctgaagcagaacac ttcagagcagtttgagacagtggcagtcaagatctttccctatgaggagt atgcctcttggaagacagagaaggacatcttctcagacatcaatctgaag catgagaacatactccagttcctgacggctgaggagcggaagacggagtt ggggaaacaatactggctgatcaccgccttccacgccaagggcaacctac aggagtacctgacgcggcatgtcatcagctgggaggacctgcgcaagctg ggcagctccctcgcccgggggattgctcacctccacagtgatcacactcc atgtgggaggcccaagatgcccatcgtgcacagggacctcaagagctcca atatcctcgtgaagaacgacctaacctgctgcctgtgtgactttgggctt tccctgcgtctggaccctactctgtctgtggatgacctggctaacagtgg gcaggtgggaactgcaagatacatggctccagaagtcctagaatccagga tgaatttggagaatgttgagtccttcaagcagaccgatgtctactccatg gctctggtgctctgggaaatgacatctcgctgtaatgcagtgggagaagt aaaagattatgagcctccatttggttccaaggtgcgggagcacccctgtg tcgaaagcatgaaggacaacgtgttgagagatcgagggcgaccagaaatt cccagcttctggctcaaccaccagggcatccagatggtgtgtgagacgtt gactgagtgctgggaccacgacccagaggcccgtctcacagcccagtgtg tggcagaacgcttcagtgagctggagcatctggacaggctctcggggagg agctgctcggaggagaagattcctgaagacggctccctaaacactaccaa atag SDI SEQ ID atggatcagacctgtgaactacccagaagaaattgtctgctgcccttttc NO: 333 caatccagtgaatttagatgcccctgaagacaaggacagccctttcggta atggtcaatccaatttttctgagccacttaatgggtgtactatgcagtta tcgactgtcagtggaacatcccaaaatgcttatggacaagattctccatc ttgttacattccactgcggagactacaggatttggcctccatgatcaatg tagagtatttaaatgggtctgctgatggatcagaatcctttcaagaccct gaaaaaagtgattcaagagctcagacgccaattgtttgcacttccttgag tcctggtggtcctacagcacttgctatgaaacaggaaccctcttgtaata actcccctgaactccaggtaaaagtaacaaagactatcaagaatggcttt ctgcactttgagaattttacttgtgtggacgatgcagatgtagattctga aatggacccagaacagccagtcacagaggatgagagtatagaggagatct ttgaggaaactcagaccaatgccacctgcaattatgagactaaatcagag aatggtgtaaaagtggccatgggaagtgaacaagacagcacaccagagag tagacacggtgcagtcaaatcgccattcttgccattagctcctcagactg aaacacagaaaaataagcaaagaaatgaagtggacggcagcaatgaaaaa gcagcccttctcccagcccccttttcactaggagacacaaacattacaat agaagagcaattaaactcaataaatttatcttttcaggatgatccagatt ccagtaccagtacattaggaaacatgctagaattacctggaacttcatca tcatctacttcacaggaattgccattttgtcaacctaagaaaaagtctac gccactgaagtatgaagttggagatctcatctgggcaaaattcaagagac gcccatggtggccctgcaggatttgttctgatccgttgattaacacacat tcaaaaatgaaagtttccaaccggaggccctatcggcagtactacgtgga ggcttttggagatccttctgagagagcctgggtggctggaaaagcaatcg tcatgtttgaaggcagacatcaattcgaagagctacctgtccttaggaga agagggaaacagaaagaaaaaggatataggcataaggttcctcagaaaat tttgagtaaatgggaagccagtgttggacttgcagaacagtatgatgttc ccaaggggtcaaagaaccgaaaatgtattcctggttcaatcaagttggac agtgaagaagatatgccatttgaagactgcacaaatgatcctgagtcaga acatgacctgttgcttaatggctgtttgaaatcactggcttttgattctg aacattctgcagatgagaaggaaaagccttgcgctaaatctcgagccaga aagagctctgataatccaaaaaggactagtgtgaaaaagggccacataca atttgaagcacataaagatgaacggaggggaaagattccagagaaccttg gcctaaactttatctctggggatatatctgatacgcaggcctctaatgaa ctttccaggatagcaaatagcctcacagggtccaacactgccccaggaag ttttctgttttcttcctgtggaaaaaacactgcaaagaaagaatttgaga cttcaaatggtgactctttattgggcttgcctgagggtgctttgatctca aagtgttctcgagagaagaataaaccccaacgaagcctggtgtgtggttc aaaagtgaagctctgctatattggagcaggtgatgaggaaaagcgaagtg attccattagtatctgtaccacttctgatgatggaagcagtgacctggat cccatagaacacagctcagagtctgataacagtgtccttgaaattccaga tgctttcgatagaacagagaacatgttatctatgcagaaaaatgaaaaga taaagtattctaggtttgctgccacaaacactagggtaaaagcaaaacag aagcctctcattagtaactcacatacagaccacttaatgggttgtactaa gagtgcagagcctggaaccgagacgtctcaggttaatctctctgatctga aggcatctactcttgttcacaaaccccagtcagattttacaaatgatgct ctctctccaaaattcaacctgtcatcaagcatatccagtgagaactcgtt aataaagggtggggcagcaaatcaagctctattacattcgaaaagcaaac agcccaagttccgaagtataaagtgcaaacacaaagaaaatccagttatg gcagaacccccagttataaatgaggagtgcagtttgaaatgctgctcttc tgataccaaaggctctcctttggccagcatttctaaaagtgggaaagtgg atggtctaaaactactgaacaatatgcatgagaaaaccagggattcaagt gacatagaaacagcagtggtgaaacatgttttatccgagttgaaggaact ctcttacagatccttaggtgaggatgtcagtgactctggaacatcaaagc catcaaaaccattacttttctcttctgcttctagtcagaatcacatacct attgaaccagactacaaattcagtacattgctaatgatgttgaaagatat gcatgatagtaagacgaaggagcagcggttgatgactgctcaaaacctgg tctcttaccggagtcctggtcgtggggactgttctactaatagtcctgta ggagtctctaaggttttggtttcaggaggctccacacacaattcagagaa aaagggagatggcactcagaactccgccaatcctagccctagtgggggtg actctgcattatctggcgagttgtctgcttccctacctggcttactgtcc gacaagagagacctccctgcttctggtaaaagtcgttcagactgtgttac taggcgcaactgtggacgatcaaagccttcatccaaattgcgagatgctt tttcagcccaaatggtaaagaacacagtgaaccgtaaagccttaaagacc gagcgcaaaagaaaactgaatcagcttccaagtgtgactcttgatgctgt actgcagggagaccgagaacgtggaggttcattgagaggtggggcagaag atcctagtaaagaggatccccttcagataatgggccacttaacaagtgaa gatggtgaccatttttctgatgtgcatttcgatagcaaggttaagcaatc tgatcctggtaaaatttctgaaaaaggactctcttttgaaaacggaaaag gcccagagctggactctgtaatgaacagtgagaatgatgaactcaatggt gtaaatcaagtggtgcctaaaaagcggtggcagcgtttaaaccaaaggcg cactaaacctcgtaagcgcatgaacagatttaaagagaaagaaaactctg agtgtgcctttagggtcttacttcctagtgaccctgtgcaggaggggcgg gatgagtttccagagcatagaactccttcagcaagcatacttgaggaacc actgacagagcaaaatcatgctgactgcttagattcagctgggccacggt taaatgtttgtgataaatccagtgccagcattggtgacatggaaaaggag ccaggaattcccagtttgacaccacaggctgagctccctgaaccagctgt gcggtcagagaagaaacgccttaggaagccaagcaagtggcttttggaat atacagaagaatatgatcagatatttgctcctaagaaaaaacaaaagaag gtacaggagcaggtgcacaaggtaagttcccgctgtgaagaggaaagcct tctagcccgaggtcgatctagtgctcagaacaagcaggtggacgagaatt ctttgatttcaaccaaagaagagcctccagttcttgaaagggaggctccg tttttggagggccccttggctcagtcagaacttggaggtggacatgctga gttgccgcagctgaccttgtctgtgcctgtggctccggaagtctctccac ggcctgcccttgagtctgaggaattgctagttaaaacgccaggaaattat gaaagtaaacgtcaaagaaaaccaactaagaaacttcttgaatccaatga tttagaccctggatttatgcccaagaagggggaccttggcctttctaaaa agtgctatgaagctggtcacctggagaatggcataactgaatcttgtgcc acatcttattcaaaagattttggtggaggcactaccaagatatttgacaa gccaaggaagcgaaaacgacagaggcatgctgcagccaagatgcagtgta aaaaagtgaaaaatgatgactcgtcaaaagagattccaggctcagaggga gaactaatgcctcacaggacggccacaagccccaaggagactgttgagga aggtgtagaacacgatcccgggatgcctgcctctaaaaaaatgcagggtg aacgcggtggaggagctgcactcaaggagaatgtctgtcagaattgtgaa aaattgggtgagctgctgttatgtgaggctcagtgctgtggggctttcca cctggagtgccttggattgactgagatgccaagaggaaaatttatctgca atgaatgtcgcacaggaatccatacctgttttgtatgtaagcagagtggg gaagatgttaaaaggtgccttctacccttgtgtggaaagttttaccatga agagtgtgtccagaagtacccacccactgttatgcagaacaagggcttcc ggtgctccctccacatctgtataacctgtcatgctgctaatccagccaat gtttctgcatctaaaggtcggttgatgcgctgtgtccgctgtcctgtggc ataccacgccaatgacttttgcctggctgctgggtcaaagatccttgcat ctaatagtatcatctgccctaatcactttacccctaggcggggctgccga aatcatgagcatgttaatgttagctggtgctttgtgtgctcagaaggagg cagccttctgtgctgtgattcttgccctgctgcttttcatcgtgaatgcc tgaacattgatatccctgaaggaaactggtattgcaatgactgtaaagca ggcaaaaagccacactacagggagattgtctgggtaaaagttggacgata caggtggtggccagctgagatctgccatcctcgagctgttccttccaaca ttgataagatgagacatgatgtgggagagttcccagtcctcttttttgga tctaatgactatttgtggactcaccaggcccgagtcttcccttacatgga gggtgacgtgagcagcaaggataagatgggcaaaggagtggatgggacat ataaaaaagctcttcaggaagctgcagcaaggtttgaggaattaaaggcc caaaaagagctaagacagctgcaggaagaccgaaagaatgacaagaagcc accaccttataaacatataaaggtaaaccgtcctattggcagggtacaga tcttcactgcagacttatctgaaataccccgttgcaactgtaaagctact gatgagaacccctgtgggatagactctgaatgcatcaaccgcatgctgct ctatgagtgccaccccacagtgtgtcctgccggagggcgctgtcaaaacc agtgcttttccaagcgccaatatccagaggttgaaattttccgcacatta cagcggggttggggtctacggacaaaaacagatattaaaaagggtgaatt tgtgaatgagtatgtgggtgagcttatagatgaagaagaatgcagagctc gaattcgctatgctcaagaacatgatatcactaatttctatatgctcacc ctagacaaagaccgaatcattgatgctggtcccaaaggaaactatgctcg gttcatgaatcattgctgccagcccaactgtgaaacacagaagtggtctg tgaatggagatacccgtgtaggcctttttgcactaagtgacattaaagca ggcactgaacttaccttcaactacaacctagaatgtcttgggaatggaaa gactgtttgcaaatgtggagccccgaactgcagtggcttcttgggtgtaa ggccaaagaatcaacccattgccacggaagaaaagtcaaagaaattcaag aagaagcaacagggaaagcgcaggacccagggtgaaatcacaaaggagcg agaagatgagtgttttagttgtggggatgctggccagctcgtctcctgca agaaaccaggctgcccaaaagtttaccacgcagactgtctcaatctgacc aagcgaccagcagggaaatgggaatgtccgtggcatcagtgtgacatctg cgggaaggaagcagcctccttctgtgagatgtgccccagctccttttgta agcagcatcgagaagggatgcttttcatttccaaactggatgggcgtctg tcttgtactgagcatgacccctgtgggcccaatcctctggaacctgggga gatccgtgagtatgtgcctcccccagtaccgctgcctccagggccaagca ctcacctggcagagcaatcaacaggaatggctgctcaggcacccaaaatg tcagataaacctcctgctgacaccaaccagatgctgtcgctctccaaaaa agctctggcagggacttgtcagaggccattgctacctgaaagacctcttg agagaactgactccaggccccagcctttagataaggtcagagacctcgct gggtcagggaccaaatcccaatccttggtttccagccagaggccactgga caggccaccagcagtggcaggaccaagaccccagctaagcgacaaaccct ctccagtgaccagcccaagctcctcaccctcagtcaggtcccaaccactg gaaagacctctggggacggctgacccaaggctggataaatccataggtgc tgccagcccaaggccccagtcactggagaaaacctcagttcccactggcc tgagacttccgccgccagacagactgctcattactagcagtcccaaaccc cagacttcagacaggcctactgacaaaccccatgcctctttgtcccagag actcccacctcctgagaaagtactatcagctgtggtccagacccttgtag ctaaagaaaaagcactgaggcctgtggaccagaatactcagtcaaaaaat agagctgctttggtgatggatctcatagacctaactcctcgccagaagga gcgggcagcttcacctcatcaggtcacaccacaggctgatgagaagatgc cagtgttggagtcaagttcatggcctgccagcaaaggtctggggcatatg ccgagagctgttgagaaaggctgtgtgtcagatcctcttcagacatctgg gaaagcagcagccccttcagaggacccctggcaagctgttaaatcactca cccaggccagacttctttctcagcctcctgccaaggcctttttatatgag ccaacaactcaggcctcaggaagagcttctgcaggggctgagcagacccc agggcctcttagccaatccccgggcctggtgaagcaggcgaagcagatgg tcggaggccagcaactacctgcacttgccgccaagagtgggcaatctttt aggtctctcgggaaggccccagcctccctccccactgaagaaaagaagtt ggtaaccacagagcaaagtccctgggccctgggaaaagcctcatcacggg cagggctctggcccatagtggctggacagacactggcacagtcttgctgg tctgctgggagcacacagacattggcacagacttgctggtctcttggaag agggcaagaccccaaaccagagcaaaatacacttccagctcttaaccagg ctccttccagtcacaagtgtgcagaatcagaacagaagtag KMT2D SEQ ID atggacagccagaagctggctggtgaggataaagattcagaaccggcagc NO: 334 tgatggacctgcagcttctgaggacccaagtgccactgagtcagacctgc ccaacccacatgtgggagaggtctctgtccttagttctgggagtcccagg cttcaggagactcctcaggactgcagtgggggtccggtgcggcgttgtgc tctctgtaactgcggggagcccagtctacacgggcagcgggagctacggc gctttgagttgccatttgattggccccggtgtccagtggtgtcccctggg gggagcccagggcccaatgaggcagtgctgcccagtgaggacctatcaca gattggtttccctgagggccttacacctgcccacctaggagaacctggag ggtcctgctgggctcaccattggtgtgctgcatggtcggcaggcgtatgg gggcaggagggcccagaactatgtggtgtggacaaggccatcttctcagg gatctcacagcgctgctcccactgcaccaggctcggtgcctccatccctt gccgctcacctggatgtccacggctttaccacttcccctgcgcgactgcc agcggttccttcctatccatgaaaacactgcagctgctatgcccagagca cagtgagggggctgcatatctggaggaggctcgctgtgcagtgtgtgagg ggccaggggagttgtgtgacctgttcttctgtaccagctgtgggcatcac tatcacggggcctgcctggacactgctctgactgcccgcaaacgtgctgg ctggcagtgccctgaatgcaaagtgtgccaagcctgcaggaaacctggga atgactctaagatgttggtttgtgagacgtgtgacaaaggataccatact ttctgcctaaaaccacccatggaggaactgcctgctcactcttggaagtg caaggcgtgccgggtgtgccgggcctgtggggcgggctcagcagaactga atcccaactcggagtggtttgagaactactctctctgtcaccgctgtcac aaagcccagggaggtcagactatccgctccgttgctgagcagcatacccc ggtgtgtagcagattttcacccccagagcctggcgatacccccactgacg agcccgatgctctgtacgttgcatgccaagggcagccaaagggtgggcac gtgacctctatgcaacccaaggaaccagggcccctgcaatgtgaagccaa accactagggaaagcaggggtccaacttgagccccagttggaggcccccc taaacgaggagatgccactgctgcccccacctgaggagtcacccctgtcc ccaccacctgaggaatcacccacgtccccaccacctgaggcatcacgcct gtcaccaccacctgaggaattgcccgcatccccacttcctgaggcattgc acctgtcccggccgctggaggaatcgcccctctctccgccgcctgaggag tctcctctgtctcccccacctgaatcatcacctttttctccactggagga gtcgcccttgtctccaccggaagagtcacccccatctcctgcacttgaga cgcctctatccccaccacctgaagcatcgcccctgtccccaccatttgaa gaatctcctttgtccccgccacctgaggaattgcccacttccccgccacc tgaagcatctcgcctgtctccaccacctgaggagtcacccatgtcccctc cacctgaagagtcacccatgtctccaccaccggaggcatctcgtctgttc ccaccatttgaagagtctcctctgtcccctccacctgaggagtctcccct ttccccaccacctgaggcatcacgcctgtccccaccacctgaggactcgc ctatgtccccaccacctgaagaatcacctatgtcccccccacctgaggta tcgcgcctatcccccctgcctgtggtgtcacgcctgtctccaccgcctga ggaatctcccttgtccccaccgcctgaggagtctcccacgtcccctccac ctgaggcttcacgcctctccccaccacctgaggactcccccacatcccca ccacctgaggactcacctgcttccccaccaccggaggactcgctcatgtc cctgccgctggaggagtcacccctgttgccactacctgaggagccgcaac tctgcccccggtccgaggggccgcacctgtcaccccggcctgaggagccg cacctgtccccccggcctgaggagccacacctatctccgcaggctgagga gccacacctgtccccccagcctgaggagccatgcctatgcgctgtgcctg aggagccacacttgtccccccaggctgagggaccacatctgtcccctcag cctgaggaattgcacctgtccccccagactgaggagccgcacctgtctcc tgtgcctgaggagccatgcttgtccccccaacctgaggaatcacacctgt ccccccagtctgaggagccatgcctgtccccccggcctgaggaatcgcat ctgtcccctgagcttgagaagccacccctgtcccctcggcctgaaaagcc ccctgaggagccaggccaatgccctgcacctgaggagctgcccttgttcc ctccccctggggaaccatccttatctcccttgcttggagagccagccctg tctgagcctggggaaccacctctgtcccctctgcccgaggagctgccgtt gtccccatctggggagccatccttgtcgcctcagctgatgccaccagatc cccttcctcctccactctcacccatcatcacagctgcggccccaccggcc ctgtctcctttgggggagttagagtacccctttggtgccaaaggggacag tgaccctgagtcaccgttggctgcccccatcctggagacacccatcagcc ctccaccagaagctaactgcactgaccctgagcctgtcccccctatgatc cttcccccatctccaggctccccagtggggccggcttctcccatcctgat ggagccccttcctcctcagtgttcgccactccttcagcattccctggttc cccaaaactcccctccttcccagtgctctcctcctgccctaccactgtcc gttccctccccgttgagtcccatagggaaggtagtgggggtctcagatga ggctgagctgcacgagatggagactgagaaagtttcagaacctgaatgcc cagccttggaacccagtgccaccagtcctctcccttccccaatgggggac ctttcctgccccgcccccagccctgccccagccctggatgacttctctgg cctaggggaagacacagcccctctggatgggattgatgctccgggttcac agccagagcctggacagacccctggcagtttggctagtgaacttaaaggc tcccctgtgctcctggaccccgaggagctggcccctgtgacccctatgga ggtctaccccgaatgcaagcagacagcagggcagggctcaccatgtgaag aacaggaagagccacgtgcaccggtggcccccacaccacccactctcatc aaatccgacatcgttaacgagatctctaatctgagccagggtgatgccag tgccagttttcctggctcagagcccctcctgggctctccagacccggagg ggggtggctccctgtccatggagttgggggtctctacggatgttagtcca gcccgagatgagggctccctacggctctgtactgactcactgccagagac tgatgactcactattgtgcgatgctgggacagctatcagcggaggcaaag ctgagggggagaaggggcggcggcgcagctccccagcccgttcccgcatc aaacagggtcgcagcagcagtttcccaggaagacgccggcctcgtggagg agcccatggaggacgtggtagaggacgggcccggctaaagtcaactgctt cttccattgagactctggtagttgctgacattgatagctctcccagtaag gaggaggaggaagaagatgatgacaccatgcagaataccgtggttctctt ctccaacacagacaaatttgtcctaatgcaggacatgtgtgtggtatgtg gcagctttggccggggggcagagggccacctccttgcctgttcgcagtgc tctcagtgctatcacccttactgtgtcaacagcaagatcaccaaggtgat gctgctcaagggctggcgttgtgtggagtgtattgtgtgtgaggtgtgtg gccaggcctccgacccctcacgcctgctgctctgtgatgactgtgatatt agctaccacacatactgcctggaccccccactgctcaccgtccccaaggg cggctggaagtgcaagtggtgtgtgtcctgtatgcagtgtggggctgctt cccctggcttccactgtgaatggcagaatagttacacacactgtgggccc tgtgccagcctggtgacctgccctatctgtcatgctccttacgtagaaga ggacctactaatccagtgccgccactgtgaacggtggatgcatgcaggct gtgagagcctcttcacagaggacgatgtggagcaggcagccgatgaaggc tttgactgtgtctcctgccagccctacgtggtaaagcctgtggcgcctgt tgcacctccagagctggtgcccatgaaggtgaaagagccagagccccagt actttcgcttcgaaggtgtgtggctgacagaaactggcatggccttgctg cgtaacctgaccatgtcaccactgcacaagcggcgccaacggcgaggacg gcttggcctcccaggcgaggcaggattggagggttctgagccctcagatg cccttggccctgatgacaagaaggatggggacctggacaccgatgagctg ctcaagggtgaaggtggtgtggagcacatggagtgcgaaattaaactgga gggccccgtcagccctgatgtggagcctggcaaagaggagaccgaggaaa gcaaaaaacgcaagcgtaaaccatatcggcctggcattggtggtttcatg gtgcgacagcggaaatcccacacacgcacgaaaaaggggcctgctgcaca ggcggaggtgttgagtggggatgggcagcccgacgaggtgatacctgctg acctgcctgcagagggcgccgtggagcagagcttagctgaaggggatgag aagaagaagcaacagcggcgagggcgcaagaagagcaaactggaggacat gttccctgcttacttgcaggaagccttctttgggaaggagctgctggacc tgagccgtaaggccctttttgcagttggggtgggccggccaagctttgga ctagggaccccaaaagccaagggagatggaggctcagaaaggaaggaact ccccacatcgcagaaaggagatgatggtccagatattgcagatgaagaat cccgtggcctcgagggcaaagccgatacaccaggacctgaggatgggggc gtgaaggcatccccagtgcccagtgaccctgagaagccaggcaccccagg tgaagggatgcttagctctgacttagacaggatttccacagaagaactgc ccaagatggaatccaaggacctgcagcagctcttcaaggatgttctgggc tctgaacgagaacagcatctgggttgtggaacccctggcctagaaggcag ccgtacgccactgcagaggccctttcttcaaggtggactccctttgggca atctgccctccagcagcccaatggactcctacccaggcctctgccagtcc ccgttcctggattctagggagcgcgggggcttctttagcccggaacccgg tgagcccgacagcccctggacgggctcaggtggcaccacgccctccaccc ccacaacccccaccacggagggtgagggcgacggactctcctataaccag cggagtcttcagcgctgggagaaggatgaggagttgggccagctgtccac catctcacctgtgctctatgccaacattaattttcctaatctcaagcaag actacccagactggtcaagccgttgcaaacaaatcatgaagctctggaga aaggttccagcagctgacaaagccccctacctgcaaaaggccaaagataa ccgggcagctcaccgcatcaacaaggtgcagaagcaggctgagagccaga tcaacaagcagaccaaggtgggcgacatagcccgtaagactgaccgaccg gccctacatctccgcattcccccgcagccaggggcactgggcagcccgcc ccccgctgctgcccccaccattttcattggcagccccactacccccgccg gcttgtctacctctgcggacgggttcctgaagccgccggcgggctcggtg cctggccctgactcgcctggtgagctcttcctcaagctcccaccccaggt gcccgcccaagtgccttcgcaggacccctttggactggcccctgcctatc ccctggagccccgcttccccacggcaccgcccacctatcccccctatcct agtcctacgggggcccctgcgcagcccccgatgctgggcgcctcatctcg tcctggggctggccagccaggggaattccacactaccccacctggcaccc ccagacaccagccctccacacctgacccattcctcaaaccccgctgcccc tcgctggataacttggctgtgcctgagagccctggggtagggggaggcaa agcttccgagcccctgctctcgcccccaccttttggggagtcccggaagg ccctagaggtgaagaaggaagagcttggggcatcctctcctagctatggg cccccaaacctgggctttgttgactcaccctcctcaggcacccacctggg tggcctggagttaaagacacctgatgtcttcaaagcccccctgacccctc gggcatctcaggtagagccccagagcccgggcttgggcctaaggccccag gagccaccccctgcccaggctttggcaccttctcctccaagtcacccaga catctttcgccctggctcctacactgacccatatgctcagcccccattga ctcctcggccccaacctccgccccctgagagctgctgtgctctgccccct cgctcactgccctccgaccctttctcccgagtgcctgccagtcctcagtc ccagtccagctcccagtctccactgacaccccggcctctgtctgctgaag ctttttgcccatcacccgttacccctcgcttccagtcccctgacccttat tctcgcccaccctcacgccctcagtcccgtgacccatttgccccattgca taagccaccccgaccccagccccctgaagttgcctttaaggctgggtctc tagcccacacttcgctgggggctggggggttcccagcagccctgcccgcg gggccagcaggtgagctccatgccaaggtcccaagtgggcagccccccaa ttttgtccggtcccctgggacgggtgcatttgtgggcaccccctctccca tgcgtttcactttccctcaggcagtaggggagccttccctaaagccccct gtccctcagcctggtctcccgccaccccatgggatcaacagccattttgg gcccggccccaccttgggcaagcctcaaagcacaaactacacagtagcca cagggaacttccacccatcgggcagccccctggggcccagcagcgggtcc acaggggagagctatgggctgtccccactacgccctccgtcggttctgcc accacctgcacccgacggatccctcccctacctgtcccatggagcctcac agcgatcaggcatcacctctcctgtcgaaaagcgagaagacccagggact ggaatgggtagctctttggcgacagctgaactcccaggtacccaggaccc aggcatgtccggccttagccaaacagagctggagaagcaacggcagcgcc agcgactacgagagctgctgattcggcagcagatccagcgcaacaccctg cggcaggagaaggaaacagctgcagcagctgcaggagcagtggggcctcc aggcagctggggtgctgagcccagcagccctgcctttgagcagctgagtc gaggccagaccccctttgctgggacacaggacaagagcagccttgtgggg ttgcccccaagcaagctgagtggccccatcctggggccagggtccttccc tagcgatgaccgactctcccggccacctccaccagccacgccttcctcta tggatgtgaacagccggcaactggtaggaggctcccaagctttctatcag cgagcaccctatcctgggtccctgcccttacagcagcaacagcaacaact gtggcagcaacaacaggcaacagcagcaacctccatgcgatttgccatgt cagctcgctttccatcaactcctggacctgaacttggccgccaagcccta ggttccccgttggcgggaatttccacccgtctgccaggccctggtgagcc agtgcctggtccagctggtcctgcccagttcattgagctgcggcacaatg tacagaaaggactgggacctgggggcactccgtttcctggtcagggccca cctcagagaccccgtttttaccctgtaagtgaggacccccaccgactggc tcctgaagggcttcggggcctggcggtatcaggtcttcccccacagaaac cctcagccccaccggcccctgaattgaacaacagtcttcatccaacaccc cacaccaagggtcctaccctgccaactggtttggagctggtcaaccggcc cccgtcgagcactgagcttggccgccccaatcctctggccctggaagctg ggaagttgccctgtgaggatcccgagctggatgacgattttgatgcccac aaggccctagaggatgatgaagagcttgctcacctgggtctgggtgtgga tgtggccaagggtgatgatgaacttggcaccttagaaaacctggagacca atgacccccacttggatgacctgctcaatggagacgagtttgacctgctg gcatatactgatcctgagctggacactggggacaagaaggatatcttcaa tgagcacctgaggctggtagaatcggctaatgagaaggctgaacgggagg ccctgctgcggggggtggagccaggacccttgggccctgaggagcgccct ccccctgctgctgatgcctctgaaccccgcctggcatctgtgctccctga ggtgaagcccaaggtggaggagggtggacgccacccttctccttgccaat tcaccattgctacccccaaggtagagcccgcacctgctgccaattccctt ggcctggggctaaagccaggacagagcatgatgggcagccgggatacccg gatgggcacagggccattttctagcagtgggcacacagctgagaaggcct cctttggggccacgggaggaccaccagctcacctgctgacccccagccca ctgagtggcccaggaggatcctccctgctggaaaagtttgagctcgagag tggggctttgaccttgcctggtggacctgcagcatctggggatgagctag acaagatggagagctcactggtagccagcgagttacccctgctcattgag gacctgttggagcatgagaagaaggagctgcagaagaagcagcagctttc agcacagttgcagcctgcccagcagcagcagcaacagcagcagcagcatt ccctactgtctgcaccaggccctgcccaggccatgtctttgccacatgag ggctcttctcccagtttggctgggtcccaacagcagctttccctgggtct tgcaggtgcccgacagccaggtttgccccagccactgatgcccacccagc caccagctcatgccctccagcaacgcctggctccatccatggctatggtg tccaatcaagggcatatgctaagtgggcagcatggagggcaggcaggctt ggtaccccagcagagctcacagccagtgctatcacagaagcccatgggca ccatgccaccttccatgtgcatgaagccgcagcaattggcaatgcagcag cagctggcaaacagcttcttcccagatacagacctggacaaatttgctgc agaagatatcattgatcccattgcaaaggccaagatggtggctttgaaag gcatcaagaaagtgatggctcagggcagcattggggtggcacctggtatg aacagacagcaagtgtctctgctagcccagaggctctcggggggacctag cagtgatctgcagaaccatgtggcagctgggagtggccaggagcggagtg ctggtgatccctcccagcctcgtcccaacccgcccacttttgctcaggga gtgatcaatgaagctgaccagcggcagtatgaggagtggctgttccatac ccagcagctcctacagatgcagctgaaggtgctagaggagcagattggtg tacaccgcaagtcccggaaggctctgtgtgccaagcagcgcactgccaaa aaagctggccgtgagttcccagaagctgatgctgagaagctcaagctggt tacagagcagcagagcaagatccagaaacaactggatcaggtccggaaac agcagaaggagcacactaatctcatggcagaatatcggaacaagcagcag caacaacagcagcagcagcagcaacaacagcaacagcactcagctgtgct ggctctcagcccttcccagagtccccggctgctcaccaagctccctggtc agctgctccctggccatgggctgcagccaccacaggggcctccgggtggg caagccggaggtcttcgcctgacccctgggggtatggcactacctggaca gcctggtggccccttccttaatacagctctggcccaacagcagcaacagc aacattctggtggggctggatccctggctggcccttcagggggcttcttc cctggcaaccttgctcttcgaagcctcggacctgattcaaggcttttaca ggaaaggcagctgcagctgcagcagcaacgtatgcagctggcccagaaac tgcagcagcagcagcagcagcaacagcagcagcagcaccttctaggacag gtggcaatccagcagcaacagcagcagggtcctggagtacagacaaacca agctctgggtcccaagccccagggccttatgcctcccagcagccaccaag gcctcctggtccagcagctgtcccctcaaccaccccaggggccccagggc atgctgggccctgcccaggtggctgtgttgcagcagcagcaccctggagc tttgggcccccagggccctcacagacaggtgcttatgacccagtcccggg tgctcagttccccccagctggcacagcagggtcagggccttatgggacac aggctggtcacagcccagcagcagcagcagcaacaacagcaccaacagca agggtccatggcagggctgtcccatcttcagcagagtctgatgtcacaca gtgggcagcccaaactgagcgctcagcccatgggctctttacagcagctt cagcagcagcagcagctgcaacagcaacagcaacttcagcagcagcagca gcagcagctacaacagcaacagcaacttcagcagcaacagcttcaacagc agcaacagcagcagcagcttcaacaacagcagcagcaacagcttcaacag cagcaacagcagctacaacagcaacagcaacaacaacagcagcagtttca acagcagcagcaacagcagcagatgggccttttaaaccagagtcgaactt tactgtctcctcagcaacaacagcagcagcaagtggcacttggccctggc atgccagcaaagcctcttcaacacttttctagccctggagccctgggtcc aaccctcctcctgacgggcaaggaacaaaacaccgtagacccagccgttt cttcagaggccactgaggggccctctacacatcagggagggccgttagca ataggaactacccctgagtcaatggccactgaaccaggagaggtaaagcc ctcactctctggggactcacaactcctgcttgtccaaccccagccccagc ctcagcccagctctctgcagctgcagccacctctgaggcttccaggacaa cagcagcagcaagttagcctgctccacacagcaggtggaggaagccatgg gcagctaggcagtggatcatcttctgaggcctcatctgtgccccacctgc tggctcagccctctgtttccttaggggatcagcctgggtccatgacccag aaccttctgggcccccaacagcccatgctagagcggcccatgcaaaataa tacagggccacaacctcccaaaccaggacctgtcctccagtctgggcagg gtctgcctggggttggaatcatgcctacggtgggtcagcttcgagcacag ctccaaggagtcctggccaaaaacccacagctgcggcacttaagtcctca gcagcagcagcagctacaggcactcctcatgcagcggcagctgcagcaga gtcaggcagtacgccagaccccaccctaccaggagcctgggacccagacc tctcccctccagggcctcctgggctgccaacctcaacttgggggcttccc tggaccacagacaggccccctccaggagctaggggcagggcctcgacctc agggcccaccccggctccctgccccaccaggagccttatctacaggacca gtccttggccctgtccatcccacacctccaccatccagccctcaagagcc aaagagaccttcacaattaccttcccccagctcccagcttcccactgagg cccagctccctcccacccatccagggacccccaaacctcaggggccaacc ttggagccgcctcctgggagggtctcacctgctgctgcccagcttgcaga taccttgtttagcaagggtctgggaccttgggatcccccagacaacctag cagaaacccagaagccagagcagagcagcctggtacctgggcatctggac caggtgaatggacaggtggtgcctgaggcatcccaactcagcatcaagca ggaacctcgggaagagccatgtgccctgggagcccagtcagtgaagaggg aggccaatggggagccaataggggcaccaggaaccagcaaccacctcctg ctggcaggccctcgctcagaagctgggcatctgctcttgcagaagctact ccgggcaaagaatgtgcaactcagcactgggcgggggtccgaggggctgc gagctgagatcaacgggcacattgacagcaagctggctgggctggagcag aaactacagggtacccccagcaacaaggaggatgcagcagcaaggaagcc tttgacaccgaagcccaagcgggtacagaaggcaagcgacaggttggtga gctcccgaaagaagctgcggaaggaggacggggtcagggccagcgaggcc ttgctgaaacagctgaaacaggagctgtccctgctgcccctaacggagcc tgctatcaccgccaattttagcctctttgccccctttggcagtggctgcc cagtcaatgggcagagccagctgaggggggcctttggaagtggggcgctg cccactggccctgactactattcccagctgcttaccaagaataacctgag taacccgccgacaccaccctcgtcgctgccccccaccccacccccatcgg tgcagcagaagatggtgaatggcgtcaccccatctgaagagctgggggag caccccaaggatgctgcctctgcccgggatagtgaaagggcactgaggga tacttcagaggtgaagagtctagacctgctggctgccttgcctacacccc ctcacaatcagactgaggatgtcaggatggagagtgatgaggatagcgat tctcctgacagcattgtgccagcttcatcccctgagagcatcttggggga ggaggcccctcgtttccctcatctgggctcaggccggtgggagcaagagg accgggccctctcccctgtcatccccctcattcctcgggccagcatccca gtcttcccagataccaaaccttatggggcccttggcctggaggtccctgg aaagctgcctgtcacaacttgggaaaagggcaaaggaagtgaggtgtcag tcatgctcacagtctctgctgctgcagccaagaacctgaatggcgtgatg gtggcagtggcggagctgctgagcatgaagatccccaactcctatgaggt gctgttcccagagagccccgcccgggcaggcactgagccaaagaaggggg aagctgagggtcctggtgggaaggaaaagggtctggaaggcaagagccca gacactggccctgattggctgaagcagtttgatgcagtgttgcctggcta taccctgaagagccaactagacatcttgagcctcctgaaacaggagagcc ccgccccagagccacccactcagcacagctatacctacaatgtctccaat ctggatgtgcgacagctctcggccccacctcctgaagaaccctccccgcc cccttcccccttggcaccttctcctgccagtccccctactgagcccttgg ttgaacttcccaccgaacccttggctgagccacccgtcccctcacctctg ccactggcctcatcccctgaatcagcccgacccaagccccgtgcccggcc ccctgaagaaggtgaagattcccgtcctcctcgcctcaagaaatggaaag gagtgcgctggaagcggcttcggctgctgctgaccatccagaagggcagt gggcggcaggaggatgagcgggaagtggcagagtttatggagcagcttgg cacagccttgcgacctgacaaggtaccgcgagacatgcgtcgctgctgtt tctgtcatgaggagggtgacggggccactgatgggcctgcccgtctgctg aacctggacctggacctgtgggtgcacctcaactgtgccctttggtccac ggaggtgtatgagacccagggcggggcactgatgaatgtggaggttgccc tgcaccgaggactgctaaccaagtgctccctgtgccagcgaactggtgcc accagcagctgcaatcgcatgcgttgccccaatgtctaccattttgcttg tgccatccgtgccaagtgcatgttcttcaaggacaagaccatgctgtgtc caatgcataagatcaaggggccctgtgagcaagagctgagctcttttgct gtcttccggcgggtctacattgagcgggacgaggtgaagcaaatcgctag catcattcagcggggagaacggctgcacatgttccgtgtggggggccttg tgttccacgccatcggacagctgctgcctcaccagatggctgactttcat agtgccactgccctctatcccgtgggctacgaggccacgcgcatctattg gagcctccgcaccaacaatcgtcgctgctgctatcgctgttctattggtg agaacaacgggcggccggagtttgtaatcaaagtcatcgagcagggcctg gaggacctggtcttcactgacgcctctccccaggccgtgtggaatcgcat cattgagcctgtggctgccatgagaaaagaggctgacatgctgcgactct tccctgagtatctgaagggcgaggagctctttgggctgacggtgcatgcc gtgcttcgcatagctgaatcactgcccggggtggagagctgtcaaaacta tttattccgctatgggcgccacccccttatggagctgccactcatgatca accccactggctgtgcccgatcagagcctaaaatcctcacacactacaaa cggccccataccctgaacagcaccagcatgtctaaggcatatcagagcac cttcacaggcgagaccaacaccccctacagcaagcagtttgtgcactcca agtcatctcagtaccggcggctgcgcaccgaatggaagaacaacgtgtac ctggctcgctcccgtatccagggcctggggctctatgcagccaaggacct agaaaagcacacaatggttatcgagtacattggcaccatcattcggaacg aggtggccaaccggcgggagaaaatctacgaagagcagaatcgaggcatc tacatgttccgaataaacaatgaacatgtgattgatgctacgttgaccgg cggccctgccaggtacattaaccattcctgtgcccctaactgtgtggccg aagtcgtgacatttgacaaagaggacaaaatcatcatcatctccagccgg cgaatccccaaaggagaggagctaacctatgactatcagtttgattttga ggacgatcagcacaagatcccctgccactgtggagcctggaattgtcgga aatggatgaactaa EP300 SEQ ID atggccgagaatgtggtggaaccggggccgccttcagccaagcggcctaa NO: 335 actctcatctccggccctctcggcgtccgccagcgatggcacagattttg gctctctatttgacttggagcacgacttaccagatgaattaatcaactct acagaattgggactaaccaatggtggtgatattaatcagcttcagacaag tcttggcatggtacaagatgcagcttctaaacataaacagctgtcagaat tgctgcgatctggtagttcccctaacctcaatatgggagttggtggccca ggtcaagtcatggccagccaggcccaacagagcagtcctggattaggttt gataaatagcatggtcaaaagcccaatgacacaggcaggcttgacttctc ccaacatggggatgggcactagtggaccaaatcagggtcctacgcagtca acaggtatgatgaacagtccagtaaatcagcctgccatgggaatgaacac agggatgaatgcgggcatgaatcctggaatgttggctgcaggcaatggac aagggataatgcctaatcaagtcatgaacggttcaattggagcaggccga gggcgacagaatatgcagtacccaaacccaggcatgggaagtgctggcaa cttactgactgagcctcttcagcagggctctccccagatgggaggacaaa caggattgagaggcccccagcctcttaagatgggaatgatgaacaacccc aatccttatggttcaccatatactcagaatcctggacagcagattggagc cagtggccttggtctccagattcagacaaaaactgtactatcaaataact tatctccatttgctatggacaaaaaggcagttcctggtggaggaatgccc aacatgggtcaacagccagccccgcaggtccagcagccaggcctggtgac tccagttgcccaagggatgggttctggagcacatacagctgatccagaga agcgcaagctcatccagcagcagcttgttctccttttgcatgctcacaag tgccagcgccgggaacaggccaatggggaagtgaggcagtgcaaccttcc ccactgtcgcacaatgaagaatgtcctaaaccacatgacacactgccagt caggcaagtcttgccaagtggcacactgtgcatcttctcgacaaatcatt tcacactggaagaattgtacaagacatgattgtcctgtgtgtctccccct caaaaatgctggtgataagagaaatcaacagccaattttgactggagcac ccgttggacttggaaatcctagctctctaggggtgggtcaacagtctgcc cccaacctaagcactgttagtcagattgatcccagctccatagaaagagc ctatgcagctcttggactaccctatcaagtaaatcagatgccgacacaac cccaggtgcaagcaaagaaccagcagaatcagcagcctgggcagtctccc caaggcatgcggcccatgagcaacatgagtgctagtcctatgggagtaaa tggaggtgtaggagttcaaacgccgagtcttctttctgactcaatgttgc attcagccataaattctcaaaacccaatgatgagtgaaaatgccagtgtg ccctccctgggtcctatgccaacagcagctcaaccatccactactggaat tcggaaacagtggcacgaagatattactcaggatcttcgaaatcatcttg ttcacaaactcgtccaagccatatttcctacgccggatcctgctgcttta aaagacagacggatggaaaacctagttgcatatgctcggaaagttgaagg ggacatgtatgaatctgcaaacaatcgagcggaatactaccaccttctag ctgagaaaatctataagatccagaaagaactagaagaaaaacgaaggacc agactacagaagcagaacatgctaccaaatgctgcaggcatggttccagt ttccatgaatccagggcctaacatgggacagccgcaaccaggaatgactt ctaatggccctctacctgacccaagtatgatccgtggcagtgtgccaaac cagatgatgcctcgaataactccacaatctggtttgaatcaatttggcca gatgagcatggcccagccccctattgtaccccggcaaacccctcctcttc agcaccatggacagttggctcaacctggagctctcaacccgcctatgggc tatgggcctcgtatgcaacagccttccaaccagggccagttccttcctca gactcagttcccatcacagggaatgaatgtaacaaatatccctttggctc cgtccagcggtcaagctccagtgtctcaagcacaaatgtctagttcttcc tgcccggtgaactctcctataatgcctccagggtctcaggggagccacat tcactgtccccagcttcctcaaccagctcttcatcagaattcaccctcgc ctgtacctagtcgtacccccacccctcaccatactcccccaagcataggg gctcagcagccaccagcaacaacaattccagcccctgttcctacacctcc tgccatgccacctgggccacagtcccaggctctacatccccctccaaggc agacacctacaccaccaacaacacaacttccccaacaagtgcagccttca cttcctgctgcaccttctgctgaccagccccagcagcagcctcgctcaca gcagagcacagcagcgtctgttcctaccccaacagcaccgctgcttcctc cgcagcctgcaactccactttcccagccagctgtaagcattgaaggacag gtatcaaatcctccatctactagtagcacagaagtgaattctcaggccat tgctgagaagcagccttcccaggaagtgaagatggaggccaaaatggaag tggatcaaccagaaccagcagatactcagccggaggatatttcagagtct aaagtggaagactgtaaaatggaatctaccgaaacagaagagagaagcac tgagttaaaaactgaaataaaagaggaggaagaccagccaagtacttcag ctacccagtcatctccggctccaggacagtcaaagaaaaagattttcaaa ccagaagaactacgacaggcactgatgccaactttggaggcactttaccg tcaggatccagaatcccttccctttcgtcaacctgtggaccctcagcttt taggaatccctgattactttgatattgtgaagagccccatggatctttct accattaagaggaagttagacactggacagtatcaggagccctggcagta tgtcgatgatatttggcttatgttcaataatgcctggttatataaccgga aaacatcacgggtatacaaatactgctccaagctctctgaggtctttgaa caagaaattgacccagtgatgcaaagccttggatactgttgtggcagaaa gttggagttctctccacagacactgtgttgctacggcaaacagttgtgca caatacctcgtgatgccacttattacagttaccagaacaggtatcatttc tgtgagaagtgtttcaatgagatccaaggggagagcgtttctttggggga tgacccttcccagcctcaaactacaataaataaagaacaattttccaaga gaaaaaatgacacactggatcctgaactgtttgttgaatgtacagagtgc ggaagaaagatgcatcagatctgtgtccttcaccatgagatcatctggcc tgctggattcgtctgtgatggctgtttaaagaaaagtgcacgaactagga aagaaaataagttttctgctaaaaggttgccatctaccagacttggcacc tttctagagaatcgtgtgaatgactttctgaggcgacagaatcaccctga gtcaggagaggtcactgttagagtagttcatgcttctgacaaaaccgtgg aagtaaaaccaggcatgaaagcaaggtttgtggacagtggagagatggca gaatcctttccataccgaaccaaagccctctttgcctttgaagaaattga tggtgttgacctgtgcttctttggcatgcatgttcaagagtatggctctg actgccctccacccaaccagaggagagtatacatatcttacctcgatagt gttcatttcttccgtcctaaatgcttgaggactgcagtctatcatgaaat cctaattggatatttagaatatgtcaagaaattaggttacacaacagggc atatttgggcatgtccaccaagtgagggagatgattatatcttccattgc catcctcctgaccagaagatacccaagcccaagcgactgcaggaatggta caaaaaaatgcttgacaaggctgtatcagagcgtattgtccatgactaca aggatatttttaaacaagctactgaagatagattaacaagtgcaaaggaa ttgccttatttcgagggtgatttctggcccaatgttctggaagaaagcat taaggaactggaacaggaggaagaagagagaaaacgagaggaaaacacca gcaatgaaagcacagatgtgaccaagggagacagcaaaaatgctaaaaag aagaataataagaaaaccagcaaaaataagagcagcctgagtaggggcaa caagaagaaacccgggatgcccaatgtatctaacgacctctcacagaaac tatatgccaccatggagaagcataaagaggtcttctttgtgatccgcctc attgctggccctgctgccaactccctgcctcccattgttgatcctgatcc tctcatcccctgcgatctgatggatggtcgggatgcgtttctcacgctgg caagggacaagcacctggagttctcttcactccgaagagcccagtggtcc accatgtgcatgctggtggagctgcacacgcagagccaggaccgctttgt ctacacctgcaatgaatgcaagcaccatgtggagacacgctggcactgta ctgtctgtgaggattatgacttgtgtatcacctgctataacactaaaaac catgaccacaaaatggagaaactaggccttggcttagatgatgagagcaa caaccagcaggctgcagccacccagagcccaggcgattctcgccgcctga gtatccagcgctgcatccagtctctggtccatgcttgccagtgtcggaat gccaattgctcactgccatcctgccagaagatgaagcgggttgtgcagca taccaagggttgcaaacggaaaaccaatggcgggtgccccatctgcaagc agctcattgccctctgctgctaccatgccaagcactgccaggagaacaaa tgcccggtgccgttctgcctaaacatcaagcagaagctccggcagcaaca gctgcagcaccgactacagcaggcccaaatgcttcgcaggaggatggcca gcatgcagcggactggtgtggttgggcagcaacagggcctcccttccccc actcctgccactccaacgacaccaactggccaacagccaaccaccccgca gacgccccagcccacttctcagcctcagcctacccctcccaatagcatgc caccctacttgcccaggactcaagctgctggccctgtgtcccagggtaag gcagcaggccaggtgacccctccaacccctcctcagactgctcagccacc ccttccagggcccccacctgcagcagtggaaatggcaatgcagattcaga gagcagcggagacgcagcgccagatggcccacgtgcaaatttttcaaagg ccaatccaacaccagatgcccccgatgactcccatggcccccatgggtat gaacccacctcccatgaccagaggtcccagtgggcatttggagccaggga tgggaccgacagggatgcagcaacagccaccctggagccaaggaggattg cctcagccccagcaactacagtctgggatgccaaggccagccatgatgtc agtggcccagcatggtcaacctttgaacatggctccacaaccaggattgg gccaggtaggtatcagcccactcaaaccaggcactgtgtctcaacaagcc ttacaaaaccttttgcggactctcaggtctcccagctctcccctgcagca gcaacaggtgcttagtatccttcacgccaacccccagctgttggctgcat tcatcaagcagcgggctgccaagtatgccaactctaatccacaacccatc cctgggcagcctggcatgccccaggggcagccagggctacagccacctac catgccaggtcagcagggggtccactccaatccagccatgcagaacatga atccaatgcaggcgggcgttcagagggctggcctgccccagcagcaacca cagcagcaactccagccacccatgggagggatgagcccccaggctcagca gatgaacatgaaccacaacaccatgccttcacaattccgagacatcttga gacgacagcaaatgatgcaacagcagcagcaacagggagcagggccagga ataggccctggaatggccaaccataaccagttccagcaaccccaaggagt tggctacccaccacagcagcagcagcggatgcagcatcacatgcaacaga tgcaacaaggaaatatgggacagataggccagcttccccaggccttggga gcagaggcaggtgccagtctacaggcctatcagcagcgactccttcagca acagatggggtcccctgttcagcccaaccccatgagcccccagcagcata tgctcccaaatcaggcccagtccccacacctacaaggccagcagatccct aattctctctccaatcaagtgcgctctccccagcctgtcccttctccacg gccacagtcccagcccccccactccagtccttccccaaggatgcagcctc agccttctccacaccacgtttccccacagacaagttccccacatcctgga ctggtagctgcccaggccaaccccatggaacaagggcattttgccagccc ggaccagaattcaatgctttctcagcttgctagcaatccaggcatggcaa acctccatggtgcaagcgccacggacctgggactcagcaccgataactca gacttgaattcaaacctctcacagagtacactagacatacactag IIRAS SEQ ID atgacggaatataagctggtggtggtgggcgccggcggtgtgggcaagag NO: 336 tgcgctgaccatccagctgatccagaaccattttgtggacgaatacgacc ccactatagaggattcctaccggaagcaggtggtcattgatggggagacg tgcctgttggacatcctggataccgccggccaggaggagtacagcgccat gcgggaccagtacatgcgcaccggggagggcttcctgtgtgtgtttgcca tcaacaacaccaagtcttttgaggacatccaccagtacagggagcagatc aaacgggtgaaggactcggatgacgtgcccatggtgctggtggggaacaa gtgtgacctggctgcacgcactgtggaatctcggcaggctcaggacctcg cccgaagctacggcatcccctacatcgagacctcggccaagacccggcag ggcagccgctctggctctagctccagctccgggaccctctgggacccccc gggacccatgtga KEAP1 SEQ ID atgcagccagatcccaggcctagcggggctggggcctgctgccgattcct NO: 337 gcccctgcagtcacagtgccctgagggggcaggggacgcggtgatgtacg cctccactgagtgcaaggcggaggtgacgccctcccagcatggcaaccgc accttcagctacaccctggaggatcataccaagcaggcctttggcatcat gaacgagctgcggctcagccagcagctgtgtgacgtcacactgcaggtca agtaccaggatgcaccggccgcccagttcatggcccacaaggtggtgctg gcctcatccagccctgtcttcaaggccatgttcaccaacgggctgcggga gcagggcatggaggtggtgtccattgagggtatccaccccaaggtcatgg agcgcctcattgaattcgcctacacggcctccatctccatgggcgagaag tgtgtcctccacgtcatgaacggtgctgtcatgtaccagatcgacagcgt tgtccgtgcctgcagtgacttcctggtgcagcagctggaccccagcaatg ccatcggcatcgccaacttcgctgagcagattggctgtgtggagttgcac cagcgtgcccgggagtacatctacatgcattttggggaggtggccaagca agaggagttcttcaacctgtcccactgccaactggtgaccctcatcagcc gggacgacctgaacgtgcgctgcgagtccgaggtcttccacgcctgcatc aactgggtcaagtacgactgcgaacagcgacggttctacgtccaggcgct gctgcgggccgtgcgctgccactcgttgacgccgaacttcctgcagatgc agctgcagaagtgcgagatcctgcagtccgactcccgctgcaaggactac ctggtcaagatcttcgaggagctcaccctgcacaagcccacgcaggtgat gccctgccgggcgcccaaggtgggccgcctgatctacaccgcgggcggct acttccgacagtcgctcagctacctggaggcttacaaccccagtgacggc acctggctccggttggcggacctgcaggtgccgcggagcggcctggccgg ctgcgtggtgggcgggctgttgtacgccgtgggcggcaggaacaactcgc ccgacggcaacaccgactccagcgccctggactgttacaaccccatgacc aatcagtggtcgccctgcgcccccatgagcgtgccccgtaaccgcatcgg ggtgggggtcatcgatggccacatctatgccgtcggcggctcccacggct gcatccaccacaacagtgtggagaggtatgagccagagcgggatgagtgg cacttggtggccccaatgctgacacgaaggatcggggtgggcgtggctgt cctcaatcgtctcctttatgccgtggggggctttgacgggacaaaccgcc ttaattcagctgagtgttactacccagagaggaacgagtggcgaatgatc acagcaatgaacaccatccgaagcggggcaggcgtctgcgtcctgcacaa ctgtatctatgctgctgggggctatgatggtcaggaccagctgaacagcg tggagcgctacgatgtggaaacagagacgtggactttcgtagcccccatg aagcaccggcgaagtgccctggggatcactgtccaccaggggagaatcta cgtccttggaggctatgatggtcacacgttcctggacagtgtggagtgtt acgacccagatacagacacctggagcgaggtgacccgaatgacatcgggc cggagtggggtgggcgtggctgtcaccatggagccctgccggaagcagat tgaccagcagaactgtacctgttga ZNF750 SEQ ID atgagtctcctcaaagagcggaagccaaaaaagccacattacatccccag NO: 338 gcctccaggaaagcccttcaagtataaatgtttccaatgtccctttactt gcaatgagaagtcacatctttttaatcacatgaagtatggtctttgtaaa aactcgattactttagtatcagagcaggatcgagttcccaagtgccctaa atctaactcactagaccccaagcaaaccaaccagcccgatgccacggcga agccagcctcttccaagtctgtcgcaaatggactctctgccttcgactcg aagcttcagcacagctctgccagggaagacatcaaggaaaacctggagct gcaagcccggggaacccacaggtgcctgggacagaagccagccctccaca gggcatcaccctgcaagagcccagctccggaagccgccctcggtgcccag cctgctctggaaggcgcagctcggccttctgcatttgttccagtcggcga gcacagactcaaggggccagacaacgccgaggcgcccgagacactggctt tacacaaccccactgccaaggccgtgtctttccacaccaagtcggccttc cacactcctggctacccctggaaagccggctcacctttccttccaccaga gtttccacataaaatctcatctacaaaggggcttggggccatttcccctt acatgcaccccacaatcccagagtacccgcctcacttttacacagagcac gggctggccaccatctactcgccttacctgctggctgggagctcgcctga gtgtgacgcacccctgctgtcagtctacggaacccaagacccgagacact tcctgcctcacccggggccgatccctaagcacctggctccatctccagcc acatacgatcactacaggtttttccagcaatatccctctaacctgccgat tccttacggattttacaggccagagtctgcattttcctcctatggtctca gactcccacctgtcactggcctcacccgagatcagagctctcacctgctt gaagaagccaccctggtctatccagcctcgagtccttccaggttaaaccc ttcggaccccaacagaaaacacgtcgagttcgaaagtccaattcctgagg ctaaagactcctccaaggctgggcagagagacacggaagggtccaaaatg agcccccgcgcagggagtgcagccacgggctccccagggaggccgagccc caccgacttcatgcagacgagccagacctgcgaaggcctgtacgacctct ccaacaaggcagcctccagcgcactgggaagactctacccgccagagcaa agcctcacagccttcaggcctgttaagaaaagcacagaatgcctacctgc ccaggctgctgagaccacagcagagtctccagtaagcctcaatgttgtga acggagaccctcctgctccgaccggaagcgcctctctcgtctcggaggcc gcgccttccagtccggacgacagctccgggatgggccccctcaacctctc caagaaatcagagataaacctggcagccacccacgaacccacgtaccaag gcagcccccaggcggaaaccgccagcttctcagagctgcaggaccttcca ctcaatctctcggtgaaggacccctgtaacacccaggctccgaggcctgc cttccccggtcgaccacgagctgcagaacctgctgctgctgttccacaga agactgggacagaaggttctgaggatgggcccagccaccctgagaccaag ccaggcagcctcgacggtgacggggccccacccacaggccccggcgagga ggctccagacgcatgcgcggtggacagcagcgaggagcagaagcagacgg cagccgtggccctgtgccagctggcggcctacagccccaggaacatccgg gtgggcgatggggatgctgcggccccggaacctgcctgccggcaagacac acccacactgagctccatggagagccaagaggcccagtgtgacctcagac ccaaaggacaaaagaggacaagtctaagggatgctggaaaatcccagcaa ggagctaagaaggcgaagctgcaggacacggccagagtgttcacactacg aaggagggcccgggtgtcctaa CCR5 SEQ ID atggattatcaagtgtcaagtccaatctatgacatcaattattatacatc NO: 339 ggagccctgccaaaaaatcaatgtgaagcaaatcgcagcccgcctcctgc ctccgctctactcactggtgttcatctttggttttgtgggcaacatgctg gtcatcctcatcctgataaactgcaaaaggctgaagagcatgactgacat ctacctgctcaacctggccatctctgacctgtttttccttcttactgtcc ccttctgggctcactatgctgccgcccagtgggactttggaaatacaatg tgtcaactcttgacagggctctattttataggcttcttctctggaatctt cttcatcatcctcctgacaatcgataggtacctggctgtcgtccatgctg tgtttgctttaaaagccaggacggtcacctttggggtggtgacaagtgtg atcacttgggtggtggctgtgtttgcgtctctcccaggaatcatctttac cagatctcaaaaagaaggtcttcattacacctgcagctctcattttccat acagtcagtatcaattctggaagaatttccagacattaaagatagtcatc ttggggctggtcctgccgctgcttgtcatggtcatctgctactcgggaat cctaaaaactctgcttcggtgtcgaaatgagaagaagaggcacagggctg tgaggcttatcttcaccatcatgattgtttattttctcttctgggctccc tacaacattgtccttctcctgaacaccttccaggaattctttggcctgaa taattgcagtagctctaacaggttggaccaagctatgcaggtgacagaga ctcttgggatgacgcactgctgcatcaaccccatcatctatgcctttgtc ggggagaagttcagaaactacctcttagtcttcttccaaaagcacattgc caaacgcttctgcaaatgctgttctattttccagcaagaggctcccgagc gagcaagctcagtttacacccgatccactggggagcaggaaatatctgtg ggcttgtga

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1. A method of genetically altering one or a plurality of primary keratinocytes comprising exposing the one or plurality of primary keratinocytes to one or a plurality of mutagens for a time period sufficient to mutate one or a plurality of wild-type genes such that one or a plurality of genome-edited primary keratinocytes deficient in expression of the one or plurality of wild-type genes are produced.
 2. (canceled)
 3. The method of claim 1, wherein the one or plurality of mutagens is a vector or an gene-modifying enzyme.
 4. (canceled)
 5. The method of claim 1 further comprising transfecting the one or plurality of primary keratinocytes with a plasmid comprising a nucleic acid sequence encoding the one or plurality of mutagens.
 6. The method of claim 5, wherein the nucleic acid sequence encodes a Cas9 protein.
 7. (canceled)
 8. The method of claim 1 further comprising a step of selecting the one or plurality of genome-edited primary keratinocytes deficient in expression of the one or plurality of wild-type genes.
 9. The method of claim 8, wherein the step of selecting is performed based on expression of a marker gene.
 10. The method of claim 9, wherein the marker gene is a fluorescent reporter gene, an antibiotic resistance gene, or a surface protein-coding gene. 11.-13. (canceled)
 14. The method of claim 1, wherein the one or plurality of genome-edited primary keratinocytes are deficient in expression of at least one of wild-type TP53, CDKN2A, and NOTCH1.
 15. The method of claim 1, wherein the one or plurality of genome-edited primary keratinocytes are deficient in expression of wild-type TP53, CDKN2A, and NOTCH1.
 16. A method of generating a population of one or a plurality of genome-edited keratinocytes comprising: a) generating and selecting one or a plurality of genome-edited primary keratinocytes deficient in expression of one or a plurality of wild-type genes according to the method of claim 1; and b) expanding the one or plurality of genome-edited primary keratinocytes into the population of one or plurality of genome-edited keratinocytes.
 17. The method of claim 16, wherein the one or plurality of primary keratinocytes are primary human keratinocytes. 18.-21. (canceled)
 22. A composition comprising one or a plurality of genome-edited keratinocytes that is deficient in expression of one or a plurality of wild-type genes chosen from TP53, CDKN2A and NOTCH1. 23.-25. (canceled)
 26. The composition of claim 22 further comprising a top layer of cells and a bottom layer of tissue, the bottom layer of tissue comprising extracellular matrix protein and one or multiple cell types and the top layer of cells comprising the one or plurality of genome-edited keratinocytes.
 27. The composition of claim 26, wherein the bottom layer of tissue comprises one or a plurality of fibroblasts.
 28. The composition of claim 26 further comprising a hydrogel layer positioned underneath the bottom layer of tissue.
 29. The composition of claim 28, wherein the hydrogel layer comprises one or a plurality of growth factors or functional fragments thereof. 30.-32. (canceled)
 33. The composition further comprising at least a single insert comprising a biocompatible polymer and an inner wall defining an inner volume divided into an upper chamber and a bottom chamber, the upper chamber and bottom chamber positioned adjacently to one another and in fluid communication and separated by a ledge; the ledge positioned along an inner wall of the insert and defining a planar interface between the upper and bottom chambers, wherein the composition comprises a top layer of cells, a bottom layer of tissue and a hydrogel; each of the top layer of cells and bottom layer of tissue positioned in the upper chamber; and wherein the hydrogel is positioned in the bottom chamber; wherein the bottom layer of tissue comprises extracellular matrix protein and the top layer of cells comprises the one or plurality of genome-edited keratinocytes keratinocytes of claim
 22. 34.-38. (canceled)
 39. The composition of claim 33, wherein the composition is frozen at from about −20 to about −212 degrees Celsius. 40.-61. (canceled)
 62. A method of isolating a primary keratinocytes from skin tissue or mucosa comprising culturing a primary keratinocyte to culture medium while simultaneously exposing one or more keratinocytes to one or a plurality of tissue samples comprising extracellular matrix (ECM).
 63. (canceled)
 64. The method of claim 62 or 63, wherein the skin is anal skin.
 65. The method of claim 62, wherein the mucosa is oral mucosa, mucosa of the upper aerodigestive tract, esophageal mucosa, vaginal mucosa, or cervical mucosa. 66.-74. (canceled)
 75. A method of screening a compound for biological activity comprising exposing one or a plurality of compounds to one or a plurality of genome-edited keratinocytes comprised in the composition of claim
 22. 76. The composition of claim 26, wherein the bottom layer of tissue comprises one or a plurality of a combination of cells including fibroblasts, endothelium, CD45+ immune cells, pericytes, and/or myoepithelial cells.
 77. (canceled) 